Registration Dossier
Registration Dossier
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EC number: - | CAS number: -
Endpoint summary
Administrative data
Key value for chemical safety assessment
Genetic toxicity in vitro
Description of key information
Three GLP, guideline compliant in vitro studies have been conducted on EC 941 -803 -4; there was no evidence of genotoxic activity in vitro in either the ames test (OECD 471), in vitro cytogenicity micronucleus assay (OECD 487) or mouse lymphoma assay (OECD 490).
As part of the read across, three in vitro studies were identified for a structurally related material, OECD 471, 487 and 490; there was no evidence of genotoxic activity in-vitro either the ames test, in vitro cytogenicity micronucleus assay or mouse lymphoma assay.
Link to relevant study records
- Endpoint:
- in vitro gene mutation study in bacteria
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 14-04-2021 to 30-04-2021
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 471 (Bacterial Reverse Mutation Assay)
- Version / remarks:
- adopted 21 July 1997, corrected 26 June 2020
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
- Version / remarks:
- August 1998
- Deviations:
- no
- GLP compliance:
- yes
- Type of assay:
- bacterial reverse mutation assay
- Target gene:
- S. typhimurium strains: his-operon
E. coli strains: tryptophan-operon - Species / strain / cell type:
- S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
- Species / strain / cell type:
- E. coli WP2 uvr A pKM 101
- Metabolic activation:
- with and without
- Metabolic activation system:
- Aroclor 1254 induced S9
- Test concentrations with justification for top dose:
- Test 1: 50, 16, 5, 1.6, 0.5, 0.16, 0.05, 0.016 mg/mL
Test 2: 100, 32, 10, 3.2, 1, 0.32, 0.1, 0.032 mg/mL
The top dose was selected based on current OECD guidelines and findings from the preliminary assessment of solubility. - Vehicle / solvent:
- Dimethyl sulfoxide (DMSO)
- Negative solvent / vehicle controls:
- yes
- Positive controls:
- yes
- Positive control substance:
- 9-aminoacridine
- 2-nitrofluorene
- sodium azide
- other: 2-Aminoanthracene; Potassium dichromate
- Details on test system and experimental conditions:
- Preliminary assessment of solubility
A preliminary solubility assessment was conducted for the test item, Petroleum Diesel/Gas Oil Fraction, Co-processed with Renewable Hydrocarbons Derived from Thermally Cracked Plastics (EC 941-803-4), at 100 mg per mL in dimethyl sulphoxide (DMSO). As the test item dissolved in DMSO at 100 mg per mL, solubility was not assessed with any other solvents and DMSO was used as the solvent for the test item throughout this study.
Mutagenicity tests
Test 1
A plate incorporation mutagenicity test was performed using Salmonella typhimurium LT2 strains TA1535, TA1537, TA98 and TA100, and Escherichia coli WP2 strain uvrA/pKM101, in both the presence and absence of S9 mix. In all cases there were three plates in the solvent control, test item and positive control groups.
Test 2
For Test 2, a liquid pre-incubation test was performed using Salmonella typhimurium LT2 strains TA1535, TA1537, TA98 and TA100, and Escherichia coli WP2 strain uvrA/pKM101 in the presence and absence of S9 mix. In all cases there were three plates in the solvent control, test item and positive control groups.
Test item administration
The test item was administered in solvent (DMSO) at a volume of 100 μL per plate for the plate incorporation method and a volume of 50 μL per plate when the liquid pre-incubation method was employed, within a maximum of two hours of formulation. - Evaluation criteria:
- A test item was considered to be mutagenic if the following criteria were satisfied:
- For all five strains, the mean number of revertant colonies is equal to or greater than 2 times the concurrent solvent control mean value at one or more doses of the test item, with or without. In addition, for TA1535 and TA1537, the mean number of revertant colonies of one or more doses of the test item, with or without metabolic activation must be equal to or greater than 2 times the relevant historical mean value.
- There was a dose-related increase in the number of revertant colonies.
- There was a reproducible (at one or more doses) increase in numbers of revertant colonies per plate in at least one strain with or without metabolic activation.
If any results had only partially satisfied the above criteria, they would be dealt with on a case-by case basis and biological relevance taken into account, for example, consistency of response within and between concentrations - Key result
- Species / strain:
- S. typhimurium TA 1535
- Remarks:
- Plate incorporation test
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- other: evidence of cytotoxicity seen at 5000 μg/plate with and without metabolic activation
- Remarks:
- precipitate was seen at and above 1600 μg per plate with and without metabolic activation
- Vehicle controls validity:
- valid
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 1537
- Remarks:
- Plate incorporation test
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- other: evidence of cytotoxicity seen at 5000 μg/plate with and without metabolic activation
- Remarks:
- precipitate was seen at and above 1600 μg per plate with and without metabolic activation
- Vehicle controls validity:
- valid
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 98
- Remarks:
- Plate incorporation test
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- other: evidence of cytotoxicity seen at 5000 μg/plate with and without metabolic activation
- Remarks:
- precipitate was seen at and above 1600 μg per plate with and without metabolic activation
- Vehicle controls validity:
- valid
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 100
- Remarks:
- Plate incorporation test
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- other: evidence of cytotoxicity seen at 5000 μg/plate with and without metabolic activation
- Remarks:
- precipitate was seen at and above 1600 μg per plate with and without metabolic activation
- Vehicle controls validity:
- valid
- Positive controls validity:
- valid
- Key result
- Species / strain:
- E. coli WP2 uvr A pKM 101
- Remarks:
- Plate incorporation test
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- other:
- Remarks:
- precipitate was seen at and above 1600 μg per plate with and without metabolic activation
- Vehicle controls validity:
- valid
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 1535
- Remarks:
- Liquid pre-incubation test
- Metabolic activation:
- with
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- other: evidence of cytotoxicity seen at 5000 μg/plate with metabolic activation; 1600 μg/plate without metabolic activation
- Remarks:
- precipitate was seen at and above 1600 μg per plate with and without metabolic activation
- Vehicle controls validity:
- valid
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 1537
- Remarks:
- Liquid pre-incubation test
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- other: evidence of cytotoxicity seen at 5000 μg/plate with metabolic activation; 1600 μg/plate without metabolic activation
- Remarks:
- precipitate was seen at and above 1600 μg per plate with and without metabolic activation
- Vehicle controls validity:
- valid
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 98
- Remarks:
- Liquid pre-incubation test
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- other: evidence of cytotoxicity seen at 5000 μg/plate with metabolic activation; 1600 μg/plate without metabolic activation
- Remarks:
- precipitate was seen at and above 1600 μg per plate with and without metabolic activation
- Vehicle controls validity:
- valid
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 100
- Remarks:
- Liquid pre-incubation test
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- other: evidence of cytotoxicity seen at 5000 μg/plate with metabolic activation; 1600 μg/plate without metabolic activation
- Remarks:
- precipitate was seen at and above 1600 μg per plate with and without metabolic activation
- Vehicle controls validity:
- valid
- Positive controls validity:
- valid
- Key result
- Species / strain:
- E. coli WP2 uvr A pKM 101
- Remarks:
- Liquid pre-incubation test
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- other: evidence of cytotoxicity seen at 5000 μg/plate without metabolic activation
- Remarks:
- precipitate was seen at and above 1600 μg per plate with and without metabolic activation
- Vehicle controls validity:
- valid
- Positive controls validity:
- valid
- Remarks on result:
- other: no mutagenic potential
- Conclusions:
- It was concluded that Petroleum Diesel/Gas Oil Fraction, Co-processed with Renewable Hydrocarbons Derived from Thermally Cracked Plastics (EC 941-803-4) was not mutagenic for Salmonella typhimurium LT2 strains TA1535, TA1537, TA98 and TA100, and Escherichia coli WP2 strain uvrA/pKM101, either in the presence or absence of S9 mix, when tested under the conditions used in this assay.
- Executive summary:
Petroleum Diesel/Gas Oil Fraction, Co-processed with Renewable Hydrocarbons Derived from Thermally Cracked Plastics (EC 941-803-4) was tested for mutagenic activity using genetically modified Salmonella typhimurium LT2 bacteria of strains TA1535, TA1537, TA98 and TA100, and Escherichia coli WP2 strain uvrA/pKM101 as indicator organisms, according to the methods of Maron and Ames, 1983, Venitt et al, 1984, Mortelmans and Zeiger, 2000 and Mortelmans and Riccio, 2000.
A preliminary solubility test was conducted. Dimethyl sulphoxide was found to be suitable and was therefore used throughout this study as the solvent for the test item.
A mutagenicity test was conducted for Petroleum Diesel/Gas Oil Fraction, Co-processed with Renewable Hydrocarbons Derived from Thermally Cracked Plastics (EC 941-803-4) using the plate incorporation method (Test 1) for all five indicator strains in both the presence and absence of an in vitro activation system based on S9 fraction obtained from Aroclor 1254-induced rat liver (S9 mix). The dose range used was 1.6 to 5000 μg per plate. As the result of Test 1 was clearly negative, a confirmatory test was carried out using the liquid pre-incubation method, Test 2, with a dose range of 1.6 to 5000 μg per plate in the presence and absence of S9 mix.
The test item showed evidence of cytotoxicity. The minimum dose level at which cytotoxicity was seen was 1600 μg per plate. The maximum dose level scored for revertant colonies was 5000 μg per plate. The minimum dose level at which precipitate was seen was 1600 μg per plate.
No significant increase in numbers of revertant (histidine or tryptophan-independent) colonies was seen with any of the five indicator strains either in the presence or absence of S9 mix.
It was concluded that Petroleum Diesel/Gas Oil Fraction, Co-processed with Renewable Hydrocarbons Derived from Thermally Cracked Plastics (EC 941-803-4) was not mutagenic for Salmonella typhimurium LT2 strains TA1535, TA1537, TA98 and TA100, and Escherichia coli WP2 strain uvrA/pKM101, either in the presence or absence of S9 mix, when tested under the conditions used in this assay.
- Endpoint:
- in vitro cytogenicity / micronucleus study
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 24 May 2021 to 15 August 2021
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)
- Version / remarks:
- 2016
- Deviations:
- not specified
- GLP compliance:
- yes (incl. QA statement)
- Type of assay:
- in vitro mammalian cell micronucleus test
- Species / strain / cell type:
- human lymphoblastoid cells (TK6)
- Metabolic activation:
- with and without
- Metabolic activation system:
- Aroclor 1254 induced S9
- Test concentrations with justification for top dose:
- The final concentration ranges tested from which micronuclei were analysed for were:
3h +S9 treatment schedule: 1481 to 3333 µg/mL
3h -S9 treatment schedule : 1481 to 3333 µg/mL
continuous treatment schedule (24h -S9): 292.6 to 987.7 µg/mL
The following levels of cytotoxicity were observed, recommended maximum level of cytotoxicity (55 ± 5%) was observed in the continuous and 3h + S9 treatment schedules:
3h +S9 treatment schedule: 57.58% at 3333 µg/mL
3h -S9 treatment schedule: 38.71% at 3333 µg/mL
continuous treatment schedule (24h -S9): 52.13% at 987.7 µg/mL
For a test item exhibiting apparent cytotoxicity, the aim was that the highest concentration selected was that which yielded cytotoxicity of ~55 ± 5%. Further doses were selected from those yielding decreasing levels of cytotoxicity, as far as a no-effect dose (little or no cytotoxicity by CBPI).
For test items that did not yield apparent cytotoxicity, the three highest test concentrations were selected for further microscopic analysis. - Vehicle / solvent:
- Dimethyl sulfoxide (DMSO)
- Negative solvent / vehicle controls:
- yes
- Positive controls:
- yes
- Positive control substance:
- colchicine
- cyclophosphamide
- mitomycin C
- Details on test system and experimental conditions:
- Test system
The study was designed to meet the requirements of OECD Guideline 487 (2016) and was agreed with the sponsor prior to commencement of the study. Since this study utilised isolated human lymphocytes and hence used cytokinesis block protocols, the specific guidance (in OECD 487) relating to lymphocytes and studies performed in the presence of cytoB, was followed.
Experimental conditions
Human lymphocytes were isolated from pooled anti-coagulated fresh blood obtained from two healthy non-smoking males and allowed to proliferate for approximately 44 to 48 hours in the presence of the mitogen, phytohaemagglutinin (PHA). Cultures of proliferating lymphocytes were exposed to the test item in the presence of S9 mix for 3 hours before cells were washed and then further incubated (humidified atmosphere of 5% CO2 at a temperature of 37oC) in fresh medium containing cytoB for approximately 21 hours. Treatments in the absence of S9 mix were performed for a 3-hour period (followed by cell washing and incubation in fresh medium containing cytoB for approximately 21 hours) and for a continuous exposure period for 24 hours. In the continuous treatment condition, cytoB was added concurrently with the test item. Duplicate cultures were treated for each test item concentration and positive control. For the solvent-treated controls, four cultures were dosed with DMSO. - Evaluation criteria:
- Criteria for a clearly positive response:
- at least one of the test concentrations exhibited a statistically significant increase compared with the concurrent negative control
- the increase was dose-related in at least one experimental condition when evaluated with an appropriate trend test
- any of the results were outside the distribution of the historical solvent control range (Poisson-based 95% control limits)
Criteria for a clearly negative response
- none of the test concentrations exhibited a statistically significant increase compared with the concurrent negative control
- there was no concentration-related increase when evaluated using an appropriate trend test
- all results were inside the distribution of the historical negative control data (Poisson-based 95% control limits)
Criteria for an equivocal response
Although most experiments are expected to yield clearly positive or negative results, in some cases the data preclude making a definitive judgement about the activity of the test item. Such equivocal responses may occur regardless of the number of times the experiment was repeated. - Statistics:
- The number of micronuclei analysed from 2000 binucleated cells for each selected test item dose was compared with that from the concurrent solvent control. Pair-wise statistical analysis employing a one-sided Fisher’s Exact test were used to evaluate statistical significance (p < 0.05). A linear trend test was employed (Cochran-Armitage) in order to confirm there was no dose related increase (p < 0.05).
- Key result
- Species / strain:
- lymphocytes: micronuclei
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity, but tested up to precipitating concentrations
- Vehicle controls validity:
- valid
- Positive controls validity:
- valid
- Remarks on result:
- other: all strains/cell types tested
- Remarks:
- no mutagenic potential
- Conclusions:
- It was concluded that Petroleum Diesel/Gas Oil Fraction, Co-processed with Renewable Hydrocarbons Derived from Thermally Cracked Plastics (EC 941-803-4) did not induce the formation of micronuclei (MN) in human lymphocytes in the absence and presence of S9, under the test conditions used. The criteria for a negative response were met.
- Executive summary:
Petroleum Diesel/Gas Oil Fraction, Co-processed with Renewable Hydrocarbons Derived from Thermally Cracked Plastics (EC 941-803-4) was tested for its potential to induce micronucleus formation in thein vitromicronucleus test with manual scoring on microscope slides. The test system used isolated human lymphocytes as the cell type and was based on methods first outlined in Fenech and Morley (1985) and later refined in Fenech (2007).
In all tests none of the treatment schedules resulted in significant increases in micronucleus formation in test item treated cultures. It was concluded that Petroleum Diesel/Gas Oil Fraction, Co-processed with Renewable Hydrocarbons Derived from Thermally Cracked Plastics (EC 941 -803 -4) did not induce the formation of the micronuclei (MN) in human lymphocytes in the absence and presence of S9, under the test conditions used.
The criteria for a negative response were met.
- Endpoint:
- in vitro gene mutation study in mammalian cells
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 10-11-2021 to 23-03-2022
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 490 (In Vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene)
- Version / remarks:
- 2016
- Deviations:
- not specified
- GLP compliance:
- yes (incl. QA statement)
- Type of assay:
- in vitro mammalian cell gene mutation tests using the thymidine kinase gene
- Target gene:
- Thymidine kinase gene
- Species / strain / cell type:
- mouse lymphoma L5178Y cells
- Remarks:
- cells sub-line Tk+/- 3.7.2C
- Details on mammalian cell type (if applicable):
- CELLS USED
- Type and source of cells: Mouse lymphoma L5178Y cells of the sub-line Tk+/- 3.7.2C (AstraZeneca Genetic Toxicology, Alderley Park (Master stock Batch: 09 September 2004))
- Suitability of cells: Thymidine kinase (TK) allows cells to salvage thymidine for use in DNA synthesis; deficiency in this enzyme is not lethal because cells are able to survive using the de novo DNA synthetic pathway. Mouse lymphoma L5178Y 3.7.2C cells are heterozygous at the Tk locus (Tk+/-) and may undergo forward mutation to the Tk-/- genotype with little or no TK activity. Both Tk+/- and Tk-/- cells are viable in non-selective medium but addition of a thymidine analogue that can be phosphorylated, e.g., trifluorothymidine (TFT) to the culture results in killing of the Tk+/- heterozygotes with preferential survival of the Tk-/- mutants. The mutant frequency can thus be estimated by comparing the cloning efficiency of cells in medium with and without the selective agent, TFT, and mutagenic activity is determined by treating cultures with different concentrations of the test substance and examining the effect on mutant frequency.
For cell lines:
- Absence of Mycoplasma contamination: Gentronix L5178Y cell stocks were checked for mycoplasma and confirmed to be mycoplasma free
- Number of passages if applicable: Cells were passaged into fresh medium every 1 to 4 days for a minimum of 4 days prior to use, following this time, cells were routinely passaged every 1 to 4 days.
- Methods for maintenance in cell culture: A vial of frozen stock L5178Y cells was removed from ultra-cold storage, thawed and initiated in culture using RPMI 1640 medium containing 10% heat-inactivated horse serum (Gibco Life Technologies, UK), antibiotics and Pluronic F68. Cells were
passaged into fresh medium every 1 to 4 days for a minimum of 4 days prior to use, following this time, cells were routinely passaged every 1 to 4 days. Incubation was at 37°C in a humidified atmosphere of 5% CO2 in air. On the day of the test, cells were counted, and the cell density adjusted to 1 x 10˄7 in 20 mL (3 hour experiments). Cells were resuspended in the appropriate media.
- Cell cycle length, doubling time or proliferation index : The doubling time for L5178Y cells is between approximately 8 and 11 hours (Lorge et al, 2016)
- Modal number of chromosomes: 40
- Periodically checked for karyotype stability: not specified
- Periodically ‘cleansed’ of spontaneous mutants: Frozen batches of cells were prepared from the master stock after purging of Tk-/- mutants by culturing for approximately 24 hours in medium containing methotrexate.
MEDIA USED
- Type and composition of media, CO2 concentration, humidity level, temperature, if applicable: For all applications, the medium was RPMI 1640 supplemented with, 2 mmol/L sodium pyruvate, 200 IU/mL penicillin and 200 μg/mL streptomycin. Pluronic F68 solution and heat-inactivated donor horse serum (HIDHS) were added to RPMI to give the different media used for various stages of the test, as follows:
Supplements Abbreviation Use
10% HIDHS R10 Stock cell maintenance
0.1% Pluronic F68 R0P Exposure to test substance (media replacement / dilution)
10% HIDHS + 0.1% Pluronic F68 R10P Exposure to test substance, maintenance during expression periods and general cell culture
20% HIDHS + 0.1% Pluronic F68 R20P Cloning cells for viability and TFT resistance - Additional strain / cell type characteristics:
- not specified
- Metabolic activation:
- with and without
- Metabolic activation system:
- Type and composition of metabolic activation system:
- source of S9 : Purchased from Molecular Toxicology Inc. (Boone, North Carolina, USA) via TRiNOVA Biochem GmbH, Germany
- method of preparation of S9 mix: Prepared from the livers of male Sprague-Dawley rats treated with Aroclor 1254. Its metabolic capacity was demonstrated by key enzyme assays and the ability to activate reference agents to bacterial mutagens. Protein content was adjusted to 30 mg/mL by dilution with RPMI 1640 before incorporation into the S9 mix used in the study. The S9 fraction was stored deep-frozen in a -80°C freezer. Sufficient vials were removed from the -80°C freezer, thawed and used; any excess S9 fraction was discarded. The complete activation system (S9 mix) was prepared immediately before use; final concentrations in the 3 hour treatment in the presence of S9 mix were as follows:
Nicotinamide adenine dinucleotide phosphate : 0.5 mM
Glucose-6-phosphate : 2.5 mM
Rat liver homogenate (S9 fraction) : 2% v/v
- quality controls of S9 (e.g., enzymatic activity, sterility, metabolic capability): conducted. - Test concentrations with justification for top dose:
- I] Range finding experiment (01): The test material was prepared in DMSO at 500.0 mg/mL. It was then prepared in a dilution series, typically with a 1.25-fold dilution factor and the nominal concentrations were:
7.206, 9.007, 11.26, 14.07, 17.59, 21.99, 27.99, 27.49, 34.36, 42.95, 53.69, 67.11, 83.89, 104.9, 131.1, 163.8, 204.8, 256.0, 320.0, 400.0 and 500.0 mg/mL (see Table 1 below)
II] Experiment 01 (3 hour treatments): In tests using 3 hours exposure, based on findings in the Range Finder Experiment 01, for the Main Experiment 01 the test material was prepared in DMSO at 67.11 mg/mL for the 3 hour -S9 treatment schedule and at 320.0 mg/mL for the 3 hour +S9 treatment schedule. It was then prepared in an appropriate dilution scheme (based upon the Range Finder results). The test material was finally diluted for treatment by adding 200 μL to 20 mL of appropriate tissue culture medium containing 1 × 10˄7 cells. The nominal concentrations of the test material were:
-S9: 15.98, 31.97, 39.96, 42.06, 44.28, 46.61, 49.06, 51.64, 54.36, 60.40 and 67.11 mg/mL
+S9: 6.880, 13.76, 27.52, 55.04, 68.80, 86.00, 107.5, 134.4, 168.0, 210.0, 233.3, 259.2, 288.0 and 320.0 mg/mL (see Table 1 below)
III] Experiment 02 (3 hour treatments): In tests using 3 hours exposure, based on findings in the Range Finder Experiment 01 and the Main Experiment 01, for the Main Experiment 02 the test item was prepared in DMSO at 67.11 mg/mL for the 3 hour –S9 treatment schedule. It was then prepared in an appropriate dilution scheme (based upon the Range Finder and Main Experiment 01 results). The test item was finally diluted for treatment by adding 200 μL to 20 mL of appropriate tissue culture medium containing 1 × 10^7 cells. The nominal concentrations of the test material were:
-S9: 15.98, 31.97, 39.96, 42.06, 44.28, 46.61, 49.06, 51.64, 54.36, 60.40 and 67.11 mg/mL (see Table 1 below) - Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: DMSO (1%)
- Justification for choice of solvent/vehicle: DMSO was selected as the test material was found to be easily soluble when diluted in it and it is a well-established solvent. The solutions were prepared on the day of use and cultures were treated within two hours of formulation.
- Justification for percentage of solvent in the final culture medium: The use of DMSO at 1% level has been shown previously to be acceptable and is customarily used in this test system. - Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- not specified
- Positive controls:
- yes
- Positive control substance:
- 4-nitroquinoline-N-oxide
- cyclophosphamide
- Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- not specified
- Positive controls:
- yes
- Positive control substance:
- other: 4-Nitroquinoline-1-oxide
- Details on test system and experimental conditions:
- NUMBER OF REPLICATIONS:
- Number of cultures per concentration (single, duplicate, triplicate) : Duplicate
- Number of independent experiments : 2 (Mutation experiment 03 and 04).
METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding (if applicable): 1 x 10˄7 in 20 mL (3 hour experiments)
- Test substance added in medium
TREATMENT AND HARVEST SCHEDULE:
- Exposure duration/duration of treatment: 3 hours
FOR GENE MUTATION:
- Expression time (cells in growth medium between treatment and selection): 48 hours
- Selection time (if incubation with a selective agent): 2 days
- Fixation time (start of exposure up to fixation or harvest of cells): 12 to 14 days
- Method used: microwell plates for the mouse lymphoma assay.
- If a selective agent is used, indicate its identity, its concentration and, duration and period of cell exposure.: Trifluorothymidine (TFT)
- Number of cells seeded and method to enumerate numbers of viable and mutants cells: 1 x 10˄7 in 20 mL (3 hour experiment).
- Criteria for small (slow growing) and large (fast growing) colonies: A colony was assessed to be large if it covered one quarter or more of the area of the well. If a well contained both a small and large colony, this was scored as a well containing a small colony.
METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method, e.g.: relative total growth (RTG)
- Any supplementary information relevant to cytotoxicity: RTG was used as the definitive measure of cytotoxicity in the mutation assay, and was the product of RSG and Relative cloning efficiency (RCE). RCE was a measure of viability at the time of selection for TFT resistant mutants in an individual culture. RCE was the CE expressed as a percentage of the mean control CE in non-selective medium (viable plates).
Relative cloning efficiency (RCE):
%RCE = (CE / Mean control CE) x 100
Relative total growth (RTG):
%RTG= (% RSG x % RCE) / 100
METHODS FOR MEASUREMENTS OF GENOTOXICITY :
Preliminary cytotoxicity test (Range finder)
In the range finder test (01), single cultures were treated with a number of concentrations of test material and the cell counts were used to calculate Relative Suspension Growth (RSG), the resulting values i.e., those best covering a cytotoxicity range (if applicable), were used to select concentrations to use on the main mutation assay. There were two solvent control cultures and no positive controls tested for each treatment. Cultures were not plated for the range finder experiment.
Mutation assay (Microwell method)
In each mutation assay (01 and 02 – 3 hour treatments), duplicate cultures were treated with a number of concentrations of test material and the most appropriate i.e., best covering a cytotoxicity range (if applicable) of 10% to 90% RSG, were sampled for CE (viability) and TFT resistance. There were four solvent control cultures and two positive control cultures tested for each treatment.
In tests using 3 hours exposure, 1 x 10^7 cells were treated in 20 mL medium containing approximately 2.5% serum.
At the end of exposure period, the cells were maintained in exponential growth for an expression period of approximately 48 hours before cloning in 96-well microtitre plates. Each culture was cloned on two selective (for TFT resistance) and two non-selective (for viability) plates with 2000 and 1.6 cells per well, respectively.
To demonstrate the mutability of the cells, 4-nitroquinoline-1-oxide (4NQO) was included as the positive control in the mutation assays without metabolic activation (-S9). Cyclophosphamide (CPA) was included in the mutation assay with metabolic activation (+S9) for the same reason and also to demonstrate the efficacy of the S9-cofactor mix. In addition, since CPA is known to cause chromosomal damage (resulting in small colonies) as well as to point mutations, its use should demonstrate the sensitivity of the test system to clastogens.
In all cases, duplicate cultures were treated with a single positive control concentration in dimethyl sulphoxide (DMSO) at a volume of 1% of the exposure medium. The use of DMSO at this level has been shown previously to be acceptable and is customarily used in this test system.
The mutant frequency (MF) for each treatment was calculated:
MF for each culture was calculated from the proportion of wells without colonies in selective medium plates, P(0)MF. It was corrected for viability using CE (above) calculated from the proportion of wells without colonies in nonselective medium, P(0)CE. Where the selective medium plates were seeded with 2000 cells per well, and the non-selective medium plates with 1.6 cells per well:
MF P (0) = (Total number of wells – number of wells with a colony) / Total number of wells
Individual MF = (−ln P(0) Mutant Plates / 2000 x (Viability (CE)) / 100)) x 1000,000
Average MF (pooled) = (MF total for that treatment condition / Total number of replicates for that treatment condition)
Induced Mutant Frequency (IMF) calculations:
Pooled IMF = Mean MF of replicate culture (test substance) - Mean MF of control
Pooled IMF small = Mean small MF of replicate culture (test substance) - Mean small IMF of control
IMF small colonies % = (IMF (small) of replicate culture (test substance) / IMF of pooled cultures (test substance)) x100 - Evaluation criteria:
- I] Criteria for conclusion of a clearly positive result:
The criteria for concluding that an individual test is clearly positive was:
a) the MF at one or more concentrations is at least 126 x 10˄-6 greater than the concurrent solvent control (GEF) and
b) there is a significant dose-relationship as indicated by trend test analysis and
c) any positive results obtained at less than 10% RTG are excluded
The positive control cultures mutant frequency values should approximate those of the acceptable ranges from the Test Facility’s historical control database.
II] Criteria for conclusion of a clearly negative result:
The criterion for concluding that an individual test is negative (considered non-mutagenic) will be:
a) The GEF is not exceeded at any test substance concentration.
b) There is no concentration related increase. A trend test will be conducted if the GEF is exceeded or if the Study Director feels that it may aid in the interpretation of a possible concentration related increase that may be just below the GEF. - Statistics:
- Please see 'Any other information on materials and methods incl. tables' for information on Statistics.'
- Key result
- Species / strain:
- mouse lymphoma L5178Y cells
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- True negative controls validity:
- not specified
- Positive controls validity:
- valid
- Additional information on results:
- TEST-SPECIFIC CONFOUNDING FACTORS
At the end of each treatment period, the osmolality and pH of the solvent control and at least the highest test concentration were determined for each treatment schedule (using an osmometer and pH meter, respectively). There were no remarkable changes observed between the solvent controls and test concentrations evaluated.
STUDY RESULTS
- Concurrent vehicle negative and positive control data
The mean solvent control (DMSO) suspension growth, cloning efficiency and mutant frequency were consistent with those stated in the OECD guideline (490) and current historical control data. The results obtained with positive control agents demonstrated the ability of the test system to identify mutagens and clastogens and where appropriate, the ability of the S9 mix to metabolise cyclophosphamide.
Treatment Schedule 1: 3 hours without S9 (-S9)
Range Finder 01
The concentration range tested was 72.06 to 5000 µg/mL. Upon addition of test item to the culture medium, precipitate was observed at a concentration of 1311 µg/mL and above. At the end of the treatment period, precipitate was observed at a concentration 5000 µg/mL. Cytotoxicity (identified as a reduction in RSG to less than 10%) was observed at a concentration of 671.1 µg/mL and above. Lower concentrations of 536.9, 429.5, 343.6, 274.9, 219.9, 175.9, 140.7, 112.6, 90.07 and 72.06 µg/mL yielded RSG values of 24, 76, 88, 94, 94, 100, 100, 94, 94 and 94%, respectively. The results obtained from the range finder experiment were used to identify suitable concentrations for the main experiment
EXPERIMENT 1:
The concentration range tested was 159.8 to 671.1 µg/mL. Upon addition of test item to the culture medium, precipitate was observed at a concentration of 399.6 µg/mL and above. At the end of the treatment period, no precipitate was observed at any concentration tested. No results will be reported for the short exposure in the absence of S9 mix for Experiment 01. Low cell counts were observed on Day 1 (24th November 2021) in doses 319.7-671.1 µg/mL. Therefore, the cultures treated with the test item in the 3 hour-S9 treatment were unsuitable for plating, as if plated it is unlikely that the RTG values would cover the cytotoxicity range, from that producing cytotoxicity and including concentrations at which there is moderate and little or no cytotoxicity. Therefore, the cultures were discarded and further testing was performed.
EXPERIMENT 2:
The concentration range tested was 159.8 to 671.1 μg/mL. Upon addition of test item to the culture medium, precipitate was observed at a concentration of 399.6 μg/mL and above. At the end of the treatment period, no precipitate was observed at any concentration tested. No results will be reported for the short exposure in the absence of S9 mix for Experiment 02. Low cell counts were observed on Day 1 (01st December 2021) in doses 319.7-671.1 μg/mL. Therefore, the cultures treated with the test item in the 3 hour-S9 treatment were unsuitable for plating, as if plated it is unlikely that the RTG values would cover the cytotoxicity range, from that producing cytotoxicity and including concentrations at which there is moderate and little or no cytotoxicity.
EXPERIMENT 3:
(Main Mutagenicity Experiment)
The concentration range tested was 17.14 to 350.0 μg/mL. Upon addition of test item to the culture medium, precipitate was observed at a concentration of 206.7 μg/mL and above. At the end of the treatment period, no precipitate was observed at any concentration tested. A mean RSG value of 2% was observed at a concentration of 350.0 μg/mL. Lower concentrations of 315.0, 283.5, 255.2, 229.6, 206.7, 186.0, 167.4, 133.9, 107.1, 85.71, 68.57, 34.28 and 17.14 μg/mL yielded mean RSG values of 2, 3, 3, 7, 8, 13, 23, 54, 88, 104, 106, 104 and 103%, respectively. The following concentrations were selected for plating 206.7, 186.0, 167.4, 133.9, 107.1 and 85.71 μg/mL.
Mean RTG values of 7 and 12% (cytotoxicity of 93 and 88%) was observed at concentrations of 206.7 and 186.0 μg/mL, respectively. Lower concentrations of 167.4, 133.9, 107.1 and 85.71 μg/mL yielded mean RTG values of 22, 49, 82 and 90%, respectively.
Data for the SG, CE and MF in the solvent control of the 3 hour treatment without S9 mix were consistent with those stated in the OECD guideline and the MF was consistent with the test facility’s historical control database (based on 95% confidence limits) for L5178Y cells and therefore, the acceptance criteria were met. Data for the small colony IMF (314x10-6) in the 4NQO positive control result of the 3 hour treatment without S9 mix were consistent with the acceptance criteria stated in the OECD guideline. The 4NQO mutant frequency was consistent with the test facility’s historical control database and a mean RTG value of 58% was observed. The mutant frequencies of the test item did not exceed the GEF at any concentrations analysed. As no concentration came close to meeting or exceeding the GEF a trend-test was not conducted as there was no apparent evidence of a relevant concentration related increase in mutant frequency. The criteria for a clear negative result were met.
Treatment Schedule 2: 3 hours with S9 (+S9)
Range Finder 01
The concentration range tested was 72.06 to 5000 μg/mL. Upon addition of test item to the culture medium, precipitate was observed at a concentration of 1311 μg/mL and above. At the end of the treatment period, precipitate was observed at a concentration 5000 μg/mL. Cytotoxicity (identified as a reduction in RSG to less than 10%) was observed at a concentration of 3200 μg/mL and above. Lower concentrations of 2560, 2048, 1638, 1311, 1049, 838.9, 671.1, 536.9, 429.5, 343.6, 274.9, 219.9, 175.9, 140.7, 112.6, 90.07 and 72.06 μg/mL yielded RSG values of 17, 44, 56, 67, 67, 72, 72, 78, 78, 83, 78, 83, 89, 89, 89, 94 and 94%, respectively.
EXPERIMENT 1:
The concentration range tested was 68.80 to 3200 μg/mL. Upon addition of test item to the culture medium, precipitate was observed at a concentration of 860.0 μg/mL and above. At the end of the treatment period, no precipitate was observed at any concentration tested. A mean RSG value of 1% was observed at a concentration of 3200 μg/mL. Lower concentrations of 2880, 2592, 2333, 2100, 1680, 1344, 1075, 860.0, 688.0, 550.4, 275.2, 137.6 and 68.80 μg/mL yielded mean RSG values of 1, 1, 2, 10, 44, 68, 81, 80, 88, 86, 87, 92 and 96%, respectively. The following concentrations were selected for plating 2100, 1680, 1344, 1075 and 137.6 μg/mL. A mean RTG of 8% (cytotoxicity of 92%) was observed at a concentration of 2100 μg/mL. Lower concentrations of 1680, 1344, 1075 and 137.6 μg/mL gave RTG values of 44, 74, 81 and 90%, respectively. However, given that the RTG for the highest concentration plated (2100 μg/mL) was 8% and the next lowest concentration (1680 μg/mL) was 44%, a concentration with an RTG between 10 and 20% was not achieved, a requirement of the OECD 490 guidelines when the maximum concentration was based on cytotoxicity. Therefore, as the acceptance criteria , further testing was performed.
EXPERIMENT 3:
The concentration range tested was 42.34 to 2500 μg/mL. Upon addition of test item to the culture medium, precipitate was observed at a concentration of 846.8 μg/mL and above. At the end of the treatment period, no precipitate was observed at any concentration tested. A mean RSG value of 1% was observed at a concentration of 2500 μg/mL. Lower concentrations of 2250, 2138, 2031, 1929, 1833, 1741, 1654, 1323, 1059, 846.8, 677.5, 338.7, 169.4, 84.68 and 42.34 μg/mL yielded mean RSG values of 2, 2, 2, 3, 3, 6, 9, 29, 55, 75, 80, 83, 78, 93 and 93%, respectively. The following concentrations were selected for plating 1654, 1323, 1059, 846.8, 338.7 and 84.68 μg/mL. A mean RTG of 8% (cytotoxicity of 92%) was observed at a concentration of 1654 μg/mL. Lower concentrations of 1323, 1059, 846.8, 338.7 and 84.68 μg/mL gave RTG values of 30, 50, 83, 86 and 91%, respectively. However, given that the RTG for the highest concentration plated (1654 μg/mL) was 8% and the next lowest concentration (1323 μg/mL) was 30%, a concentration with an RTG between 10 and 20% was not achieved, a requirement of the OECD 490 guidelines when the maximum concentration was based on cytotoxicity. Therefore, as the acceptance criteria , further testing was performed.
EXPERIMENT 4:
(Main Mutagenicity Experiments)
The concentration range tested was 43.11 to 2250 μg/mL. Upon addition of test item to the culture medium, precipitate was observed at a concentration of 689.7 μg/mL and above. At the end of the treatment period, no precipitate was observed at any concentration tested.
A mean RSG value of 2% was observed at a concentration of 2250 μg/mL. Lower concentrations of 2138, 2031, 1929, 1833, 1741, 1654, 1571, 1493, 1418, 1347, 1078, 862.2, 689.7, 344.9, 172.4, 86.22 and 43.11 μg/mL yielded mean RSG values of 18, 4, 6, 5, 6, 12, 8, 11, 14, 18, 31, 38, 43, 51, 76, 81 and 97%, respectively. The following concentrations were selected for plating 1654, 1493, 1418, 1347, 1078, 862.2, 344.9, 172.4 and 43.11 μg/mL.
Mean RTG values of 11, 10, 12 and 15% (cytotoxicity of 89, 90, 88 and 85%) was observed at concentrations of 1654, 1493, 1418 and 1347 μg/mL, respectively. Lower concentrations of 1078, 862.2, 344.9, 172.4 and 43.11 μg/mL gave RTG values of 33, 34, 57, 69 and 88%, respectively. However, a high CE value for one of the 1418 μg/mL cultures was recorded, this was thought to be due to a sampling error when preparing the cell suspension for plating. Therefore, this culture has been excluded from the data analysis; this is deemed as acceptable as single cultures can be dosed in accordance with the OECD guidelines and will not impact the validity and integrity of the study.
Data for the SG, CE and MF in the solvent control of the 3 hour treatment with S9 mix were consistent those stated in the OECD guideline and the MF was consistent with the test facility’s historical control database (based on 95% confidence limits) for L5178Y cells and therefore, the acceptance criteria were met. Data for the IMF (766x10-6), small colony IMF (337x10-6) and IMF small colonies percentage (44%) in the CPA positive control result of the 3 hour with S9 mix were consistent with the acceptance criteria stated in the OECD guideline. The CPA mutant frequency was consistent with the test facility’s historical control database and a mean RTG value of 70% was observed. The mutant frequencies of the test item did not exceed the GEF at any concentrations analysed. As no concentration came close to meeting or exceeding the GEF a trend-test was not conducted as there was no apparent evidence of a relevant concentration related increase in mutant frequency. The criteria for a clear negative result were met.
There was no indication of a requirement to conduct testing for 24 hours without S9, as outlined in the OECD guideline. Namely, the test item was readily soluble and is not known to be of a class of substances that require longer exposure times to be detected (e.g. a nucleoside analogue). Further testing with a 24 hours without S9 treatment schedule was not required for this test item, this has no impact on the validity and integrity of this study.
HISTORICAL CONTROL DATA (with ranges, means and standard deviation, and 95% control limits for the distribution as well as the number of data)
- Positive historical control data:
Please see 'Table 4' in 'Any other information on results incl. tables' for information on historical control data.
- Negative (solvent/vehicle) historical control data: Please see 'Table 3' in 'Any other information on results incl. tables' for information on historical control data. - Conclusions:
- It was concluded that Petroleum Diesel/Gas Oil Fraction, Co-processed with Renewable Hydrocarbons Derived from Thermally Cracked Plastics (EC 941-803-4) was not mutagenic in the mouse lymphoma L5178Y Tk locus assay in the absence and presence of S9 mix, and under the test conditions used, the criteria for a clear negative response were met.
- Executive summary:
Petroleum Diesel/Gas Oil Fraction, Co-processed with Renewable Hydrocarbons Derived from Thermally Cracked Plastics (EC 941-803-4) was tested for its potential to induce forward mutation at the thymidine kinase (Tk) locus in mouse lymphoma L5178Y Tk+/- -3.7.2C cells.
Petroleum Diesel/Gas Oil Fraction, Co-processed with Renewable Hydrocarbons Derived from Thermally Cracked Plastics (EC 941-803-4) was applied to the test system under two treatment schedules in accordance with the OECD Guideline 490 (2016): treatment for 3 hours in both the absence and presence of an in vitro activation system based on S9 fraction obtained from Aroclor 1254-induced rat liver (S9 mix). In all treatments, the solvent used for the test item was dimethyl sulphoxide (DMSO). The microwell method of the mouse lymphoma test was used for the mutation test.
The final concentration ranges that were plated and analysed for cloning efficiency (CE) and TFT resistance, which met the acceptance criteria were:
3h –S9 mix treatment schedule: 85.71 to 206.7 μg/mL (Experiment 03)
3h +S9 mix treatment schedule: 43.11 to 1654 μg/mL (Experiment 04)
Data for the suspension growth (SG), CE and Mutant Frequency (MF) in the solvent control for all treatment schedules were consistent with those stated in the OECD guideline and the MF was consistent with the test facility’s historical control database (based on 95% confidence limits) for L5178Y cells and therefore, the acceptance criteria were met.
The positive control results for all treatment schedules were consistent with the acceptance criteria stated in the OECD guideline and the MF was consistent with the test facility’s historical control database (based on 95% confidence limits) for L5178Y cells.
In Experiment 03 the 3 hour –S9 treatment exposure concentrations of 206.7 and 186.0 μg/mL gave 7 and 12% relative total growth (RTG) (cytotoxicity of 93 and 88%), respectively. In Experiment 04 the 3 hour +S9 treatment exposure concentrations of 1654, 1493, 1418 and 1347 μg/mL gave 11, 10, 12 and 15% RTG (cytotoxicity of 89, 90, 88 and 85%), respectively.
In final, valid mutation assay experiments, upon addition of the test item to the culture medium, precipitate was observed at a concentration of 206.7 μg/mL and above in the 3h –S9 mix treatment schedule (Experiment 03) and at a concentration of 689.7 μg/mL and above in the 3h +S9 mix treatment schedule (Experiment 04). At the end of the treatment period, no precipitate was observed in either treatment schedule.
There were no relevant changes in MF for any of the treatment schedules at any of the concentrations analysed. The Global Evaluation Factor (GEF) was not exceeded for any of the analysed test concentrations (which met the acceptance criteria). The criteria for a clear negative response were met for all treatment schedules.
It was concluded that Petroleum Diesel/Gas Oil Fraction, Co-processed with Renewable Hydrocarbons Derived from Thermally Cracked Plastics (EC 941-803-4) was not mutagenic in the mouse lymphoma L5178Y Tk locus assay in the absence and presence of S9 mix, and under the test conditions used, the criteria for a clear negative response were met.
- Endpoint:
- in vitro gene mutation study in bacteria
- Remarks:
- Type of genotoxicity: gene mutation
- Type of information:
- experimental study
- Adequacy of study:
- supporting study
- Study period:
- 09-Nov-2020 to 20-Nov-2020
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 471 (Bacterial Reverse Mutation Assay)
- Qualifier:
- according to guideline
- Guideline:
- EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
- Qualifier:
- according to guideline
- Guideline:
- EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
- GLP compliance:
- yes (incl. QA statement)
- Type of assay:
- bacterial reverse mutation assay
- Target gene:
- S. typhimurium strains: his-operon
E. coli strains: tryptophan-operon - Species / strain / cell type:
- S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
- Species / strain / cell type:
- E. coli WP2 uvr A pKM 101
- Metabolic activation:
- with and without
- Metabolic activation system:
- Aroclor 1254 induced S9
- Vehicle / solvent:
- Dimethyl sulfoxide (DMSO)
- Negative solvent / vehicle controls:
- yes
- Positive controls:
- yes
- Positive control substance:
- 9-aminoacridine
- 2-nitrofluorene
- sodium azide
- other: 2-aminoanthracene, Potassium dichromate
- Details on test system and experimental conditions:
- Preliminary assessment of solubility
A preliminary solubility assessment was conducted for the test item, Petroleum Gas Oil Fraction, Co-processed with Renewable Hydrocarbons of Plant and/or Animal Origin (EC 941-364-9), at 100 mg per mL in dimethyl sulphoxide (DMSO). As the test item dissolved in DMSO at 100 mg per mL, solubility was not assessed with any other solvents and DMSO was used as the solvent for the test item throughout this study.
Mutagenicity tests
Test 1
A plate incorporation mutagenicity test was performed using Salmonella typhimurium LT2 strains TA1535, TA1537, TA98 and TA100, and Escherichia coli WP2 strain uvrA/pKM101, in both the presence and absence of S9 mix. In all cases there were three plates in the solvent control, test item and positive control groups.
Test 2
For Test 2, a liquid pre-incubation test was performed using Salmonella typhimurium LT2 strains TA1535, TA1537, TA98 and TA100, and Escherichia coli WP2 strain uvrA/pKM101 in the presence and absence of S9 mix. In all cases there were three plates in the solvent control, test item and positive control groups.
Test item administration
The test item was administered in solvent (DMSO) at a volume of 100 μL per plate for the plate incorporation method and a volume of 50 μL per plate when the liquid pre-incubation method was employed, within a maximum of three hours of formulation. - Evaluation criteria:
- Test acceptance criteria
A minimum of five analysable (scoreable) concentrations was required, with at least four showing no signs of cytotoxicity.
Evaluation
Cytotoxicity
A dose of the test item was judged to be toxic to a bacterial strain if the formation of microcolonies (background lawn) was reduced, or a relevant decrease in the number of revertant colonies was seen.
Mutagenicity
A test item was considered to be mutagenic if the following criteria were satisfied:
- For all five strains, the mean number of revertant colonies is equal to or greater than 2 times the concurrent solvent control mean value at one or more doses of the test item, with or without. In addition, for TA1535 and TA1537, the mean number of revertant colonies of one or more doses of the test item, with or without metabolic activation must be equal to or greater than 2 times the relevant historical mean value.
- There was a dose-related increase in the number of revertant colonies.
- A reproducible (at one or more doses) increase in numbers of revertant colonies per plate in at least one strain with or without metabolic activation. - Statistics:
- The mean number of revertant colonies and standard deviations were calculated for all groups.
All valid data were plotted and analysed using a linear regression analysis programme. - Key result
- Species / strain:
- S. typhimurium TA 1535
- Remarks:
- Plate incorporation test
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- other: above 5000 μg/plate with metabolic activation
- Vehicle controls validity:
- valid
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 1537
- Remarks:
- Plate incorporation test
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- other: above 5000 μg/plate with metabolic activation
- Vehicle controls validity:
- valid
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 98
- Remarks:
- Plate incorporation test
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- other: above 5000 μg/plate with metabolic activation
- Vehicle controls validity:
- valid
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 100
- Remarks:
- Plate incorporation test
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- other: above 5000 μg/plate with metabolic activation
- Vehicle controls validity:
- valid
- Positive controls validity:
- valid
- Key result
- Species / strain:
- E. coli WP2 uvr A pKM 101
- Remarks:
- Plate incorporation test
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- not specified
- Vehicle controls validity:
- valid
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 1535
- Remarks:
- Liquid pre-incubation test
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- above 500 μg/plate without metabolic activation; 1600 μg/plate with metabolic activation
- Vehicle controls validity:
- valid
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 1537
- Remarks:
- Liquid pre-incubation test
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- above 500 μg/plate without metabolic activation; 1600 μg/plate with metabolic activation
- Vehicle controls validity:
- valid
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 98
- Remarks:
- Liquid pre-incubation test
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- above 500 μg/plate without metabolic activation; 1600 μg/plate with metabolic activation
- Vehicle controls validity:
- valid
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 100
- Remarks:
- Liquid pre-incubation test
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- above 500 μg/plate without metabolic activation; 1600 μg/plate with metabolic activation
- Vehicle controls validity:
- valid
- Positive controls validity:
- valid
- Key result
- Species / strain:
- E. coli WP2 uvr A pKM 101
- Remarks:
- Liquid pre-incubation test
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- 5000 μg/plate with metabolic activation
- Vehicle controls validity:
- valid
- Positive controls validity:
- valid
- Additional information on results:
- Plate incorporation test
On the day of dosing, precipitate was seen at and above 500 μg per plate for all five strains in both the presence and absence of S9 mix. On the day of scoring, precipitate was seen at and above 1600 μg per plate for all five strains in both the presence and absence of S9 mix.
Evidence of cytotoxicity as indicated by reductions in the growth of the background lawns or in the incidence of spontaneous revertant colonies was seen at a dose of 5000 μg per plate for the four S. typhimurium strains in the absence of S9 mix.
No significant increase in numbers of revertant (histidine or tryptophan-independent) colonies was seen with any of the five indicator strains either in the presence or absence of S9 mix.
Liquid pre-incubation test
On the day of dosing precipitate was seen at and above 500 μg per plate for all five strains in both the presence and absence of S9 mix. On the day of scoring, precipitate was seen at and above 1600 μg per plate for all five strains in both the presence and absence of S9 mix.
Evidence of cytotoxicity as indicated by reductions in the growth of the background lawns or in the incidence of spontaneous revertant colonies was seen at and above a dose of 500 μg per plate for the four S. typhimurium strains in the absence of S9 mix, at and above 1600 μg per plate for four S. typhimurium strains in the presence of S9 mix, and at 5000 μg per plate for the E. coli strain in the absence of S9 mix.
No significant increase in numbers of revertant (histidine or tryptophan-independent) colonies was seen with any of the five indicator strains either in the presence or absence of S9 mix. - Remarks on result:
- other: all strains/cell types tested
- Remarks:
- no mutagenic potential
- Conclusions:
- It was concluded that Petroleum Gas Oil Fraction, Co-processed with Renewable Hydrocarbons of Plant and/or Animal Origin (EC 941-364-9) was not mutagenic for Salmonella typhimurium LT2 strains TA1535, TA1537, TA98 and TA100, and Escherichia coli WP2 strain uvrA/pKM101, either in the presence or absence of S9 mix, when tested under the conditions used in this assay.
- Executive summary:
Petroleum Gas Oil Fraction, Co-processed with Renewable Hydrocarbons of Plant and/or Animal Origin (EC 941-364-9) was tested for mutagenic activity using genetically modified Salmonella typhimurium LT2 bacteria of strains TA1535, TA1537, TA98 and TA100, and Escherichia coli WP2 strain uvrA/pKM101 as indicator organisms, according to the methods of Maron and Ames, 1983, Venitt et al, 1984, Mortelmans and Zeiger, 2000 and Mortelmans and Riccio, 2000.
A preliminary solubility test was conducted. Dimethyl sulphoxide was found to be suitable and was therefore used throughout this study as the solvent for the test item.
A mutagenicity test was conducted for Petroleum Gas Oil Fraction, Co-processed with Renewable Hydrocarbons of Plant and/or Animal Origin (EC 941-364-9) using the plate incorporation method (Test 1) for all five indicator strains in both the presence and absence of an in vitro activation system based on S9 fraction obtained from Aroclor 1254-induced rat liver (S9 mix). The dose range used was 1.6 to 5000 μg per plate. As the result of Test 1 was clearly negative, a confirmatory test was carried out using the liquid pre-incubation method, Test 2, using a dose range of 1.6 to 5000 μg per plate in the presence and absence of S9 mix.
The test item showed evidence of cytotoxicity. The minimum dose level at which cytotoxicity was seen was 500 μg per plate. The maximum dose level scored for revertant colonies was 5000 μg per plate. The minimum dose level at which precipitate was seen on the test plates was 1600 μg per plate.
No significant increase in numbers of revertant (histidine or tryptophan-independent) colonies was seen with any of the five indicator strains either in the presence or absence of S9 mix.
It was concluded that Petroleum Gas Oil Fraction, Co-processed with Renewable Hydrocarbons of Plant and/or Animal Origin (EC 941-364-9) was not mutagenic for Salmonella typhimurium LT2 strains TA1535, TA1537, TA98 and TA100, and Escherichia coli WP2 strain uvrA/pKM101, either in the presence or absence of S9 mix, when tested under the conditions used in this assay.
- Endpoint:
- in vitro gene mutation study in mammalian cells
- Type of information:
- experimental study
- Adequacy of study:
- supporting study
- Study period:
- 2021-AUG-19 to 2021-NOV-24
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 490 (In Vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene)
- Version / remarks:
- 2016
- Deviations:
- not specified
- GLP compliance:
- yes (incl. QA statement)
- Type of assay:
- in vitro mammalian cell gene mutation tests using the thymidine kinase gene
- Target gene:
- Thymidine kinase gene
- Species / strain / cell type:
- mouse lymphoma L5178Y cells
- Remarks:
- cells sub-line Tk+/- 3.7.2C
- Details on mammalian cell type (if applicable):
- CELLS USED
- Type and source of cells: Mouse lymphoma L5178Y cells of the sub-line Tk+/- 3.7.2C (AstraZeneca Genetic Toxicology, Alderley Park (Master stock Batch: 09 September 2004))
- Suitability of cells: Thymidine kinase (TK) allows cells to salvage thymidine for use in DNA synthesis; deficiency in this enzyme is not lethal because cells are able to survive using the de novo DNA synthetic pathway. Mouse lymphoma L5178Y 3.7.2C cells are heterozygous at the Tk locus (Tk+/-) and may undergo forward mutation to the Tk-/- genotype with little or no TK activity. Both Tk+/- and Tk-/- cells are viable in non-selective medium but addition of a thymidine analogue that can be phosphorylated, e.g., trifluorothymidine (TFT) to the culture results in killing of the Tk+/- heterozygotes with preferential survival of the Tk-/- mutants. The mutant frequency can thus be estimated by comparing the cloning efficiency of cells in medium with and without the selective agent, TFT, and mutagenic activity is determined by treating cultures with different concentrations of the test substance and examining the effect on mutant frequency.
For cell lines:
- Absence of Mycoplasma contamination: Gentronix L5178Y cell stocks were checked for mycoplasma and confirmed to be mycoplasma free
- Number of passages if applicable: Cells were passaged into fresh medium every 1 to 4 days for a minimum of 4 days prior to use, following this time, cells were routinely passaged every 1 to 4 days.
- Methods for maintenance in cell culture: A vial of frozen stock L5178Y cells was removed from ultra-cold storage, thawed and initiated in culture using RPMI 1640 medium containing 10% heat-inactivated horse serum (Gibco Life Technologies, UK), antibiotics and Pluronic F68. Cells were passaged into fresh medium every 1 to 4 days for a minimum of 4 days prior to use, following this time, cells were routinely passaged every 1 to 4 days. Incubation was at 37°C in a humidified atmosphere of 5% CO2 in air. On the day of the test, cells were counted, and the cell density adjusted to 1 x 10˄7 in 20 mL (3 hour experiments). Cells were resuspended in the appropriate media.
- Cell cycle length, doubling time or proliferation index: The doubling time for L5178Y cells is between approximately 8 and 11 hours (Lorge et al, 2016).
- Modal number of chromosomes: 40
- Periodically checked for karyotype stability: not specified
- Periodically ‘cleansed’ of spontaneous mutants: Frozen batches of cells were prepared from the master stock after purging of Tk-/- mutants by culturing for approximately 24 hours in medium containing methotrexate.
MEDIA USED
- Type and composition of media, CO2 concentration, humidity level, temperature, if applicable: For all applications, the medium was RPMI 1640 supplemented with, 2 mmol/L sodium pyruvate, 200 IU/mL penicillin and 200 μg/mL streptomycin. Pluronic F68 solution and heat-inactivated donor horse serum (HIDHS) were added to RPMI to give the different media used for various stages of the test, as follows:
Supplements Abbreviation Use
10% HIDHS R10 Stock cell maintenance
0.1% Pluronic F68 R0P Exposure to test substance (media replacement / dilution)
10% HIDHS + 0.1% Pluronic F68 R10P Exposure to test substance, maintenance during expression periods and general cell culture
20% HIDHS + 0.1% Pluronic F68 R20P Cloning cells for viability and TFT resistance - Additional strain / cell type characteristics:
- not specified
- Metabolic activation:
- with and without
- Metabolic activation system:
- Type and composition of metabolic activation system:
- source of S9: Purchased from Molecular Toxicology Inc. (Boone, North Carolina, USA) via TRiNOVA Biochem GmbH (Germany)
- method of preparation of S9 mix: Prepared from the livers of male Sprague-Dawley rats treated with Aroclor 1254. Its metabolic capacity was demonstrated by key enzyme assays and the ability to activate reference agents to bacterial mutagens. Protein content was adjusted to 30 mg/mL by dilution with RPMI 1640 before incorporation into the S9 mix used in the study. The S9 fraction was stored deep-frozen in a -80°C freezer. Sufficient vials were removed from the -80°C freezer, thawed and used; any excess S9 fraction was discarded. The complete activation system (S9 mix) was prepared immediately before use; final concentrations in the 3 hour treatment in the presence of S9 mix were as follows:
Nicotinamide adenine dinucleotide phosphate : 0.5 mM
Glucose-6-phosphate : 2.5 mM
Rat liver homogenate (S9 fraction): 2% v/v
- concentration or volume of S9 mix and S9 in the final culture medium: 2% v/v (final concentration in the 3 hour treatment in the presence of S9 mix)
- quality controls of S9 (e.g., enzymatic activity, sterility, metabolic capability): Not specified - Test concentrations with justification for top dose:
- I] Range finding experiment (01): The test material was prepared in DMSO at 150.0 mg/mL. It was then prepared in a dilution series, typically with a 1.25-fold dilution factor and the nominal concentrations were:
2.162, 2.702, 3.378, 4.222, 5.278, 6.597, 8.246, 10.31, 12.88, 16.11, 20.13, 25.17, 31.46, 39.32, 49.15, 61.44, 76.80, 96.00, 120.0, and 150.0 mg/mL
II] Mutation Experiment 01 (3-hour treatments): Based on findings in the Range Finder Experiment 01, for the Main Experiment 01 the test material was prepared in DMSO at 31.46 mg/mL for the 3 hour -S9 treatment schedule and at 150.0 mg/mL for the 3 hour +S9 treatment schedule. It was then prepared in an appropriate dilution scheme (based upon the Range Finder results). The test material was finally diluted for treatment by adding 200 μL to 20 mL of appropriate tissue culture medium containing 1 × 10˄7 cells. The nominal concentrations of the test material were:
-S9: 3.762, 7.524, 15.05, 16.72, 18.58, 20.64, 22.93, 25.48, 28.31, and 31.46 mg/mL
+S9: 1.920, 3.840, 7.680, 15.36, 30.72, 61.44, 76.80, 96.00, 108.0, 120.0, and 150.0 mg/mL
III] Mutation Experiment 02 (3-hour treatments): Based on findings in the Range Finder Experiment 01 and the Main Experiment 01, for the Main Experiment 02 the test material was prepared in DMSO at 31.46 mg/mL for the 3 hour –S9 treatment schedule and at 150.0 mg/mL for the 3 hour +S9 treatment schedule was then prepared in an appropriate dilution scheme (based upon the Range Finder and Main Experiment 01 results). The test material was finally diluted for treatment by adding 200 μL to 20 mL of appropriate tissue culture medium containing 1 × 10˄7 cells. The nominal concentrations of the test material were:
-S9: 3.998, 7.997, 8.885, 9.872, 10.97, 12.19, 13.54, 15.05, 16.72, 18.58, 20.64, 22.93, 25.48, 28.31 and 31.46 mg/mL
+S9: 1.920, 3.840, 7.680, 15.36, 30.72, 61.44, 76.80, 96.00, 108.3, 114.0, 120.0 and 150.0 mg/mL - Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: Dimethyl sulfoxide DMSO (1%)
- Justification for choice of solvent/vehicle: DMSO was selected as the test material was found to be easily soluble when diluted in it and it is a well-established solvent. The solutions were prepared on the day of use and cultures were treated within two hours of formulation.
- Justification for percentage of solvent in the final culture medium: Not specified - Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- not specified
- Positive controls:
- yes
- Positive control substance:
- 4-nitroquinoline-N-oxide
- cyclophosphamide
- Details on test system and experimental conditions:
- NUMBER OF REPLICATIONS:
- Number of cultures per concentration (single, duplicate, triplicate) : Duplicate
- Number of independent experiments : 2 (Mutation experiment 01 and 02)
METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding (if applicable): 1 x 10˄7 in 20 mL (3 hour experiments)
- Test substance added in medium
TREATMENT AND HARVEST SCHEDULE:
- Exposure duration/duration of treatment: 3 hours
FOR GENE MUTATION:
- Expression time (cells in growth medium between treatment and selection): 48 hours
- Selection time (if incubation with a selective agent): 2 days
- Fixation time (start of exposure up to fixation or harvest of cells): 12 to 14 days
- Method used: microwell plates for the mouse lymphoma assay.
- If a selective agent is used, indicate its identity, its concentration and, duration and period of cell exposure.: Trifluorothymidine (TFT)
- Number of cells seeded and method to enumerate numbers of viable and mutants cells: 1 x 10˄7 in 20 mL (3 hour experiment).
- Criteria for small (slow growing) and large (fast growing) colonies: A colony was assessed to be large if it covered one quarter or more of the area of the well. If a well contained both a small and large colony, this was scored as a well containing a small colony.
METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method, e.g.: relative total growth (RTG)
- Any supplementary information relevant to cytotoxicity: RTG was used as the definitive measure of cytotoxicity in the mutation assay, and was the product of RSG and Relative cloning efficiency (RCE). RCE was a measure of viability at the time of selection for TFT resistant mutants in an individual culture. RCE was the CE expressed as a percentage of the mean control CE in non-selective medium (viable plates).
Relative cloning efficiency (RCE):
%RCE = (CE / Mean control CE) x 100
Relative total growth (RTG):
%RTG= (% RSG x % RCE) / 100
METHODS FOR MEASUREMENTS OF GENOTOXICITY :
Preliminary cytotoxicity test (Range finder)
In the range finder test (01), single cultures were treated with a number of concentrations of test material and the cell counts were used to calculate Relative Suspension Growth (RSG), the resulting values i.e., those best covering a cytotoxicity range (if applicable), were used to select concentrations to use on the main mutation assay. There were two solvent control cultures and no positive controls tested for each treatment. Cultures were not plated for the range finder experiment.
Mutation assay (Microwell method)
In each mutation assay (01 and 02 – 3 hour treatments), duplicate cultures were treated with a number of concentrations of test material and the most appropriate i.e., best covering a cytotoxicity range (if applicable) of 10% to 90% RSG, were sampled for CE (viability) and TFT resistance. There were four solvent control cultures and two positive control cultures tested for each treatment. In tests using 3 hours exposure, 1 x 10˄7 cells were treated in 20 mL medium containing approximately 2.5% serum.
At the end of exposure period, the cells were maintained in exponential growth for an expression period of approximately 48 hours before cloning in 96-well microtitre plates. Each culture was cloned on two selective (for TFT resistance) and two non-selective (for viability) plates with 2000 and 1.6 cells per well, respectively.
To demonstrate the mutability of the cells, 4-nitroquinoline-1-oxide (4NQO) was included as the positive control in the mutation assays without metabolic activation (-S9). Cyclophosphamide (CPA) was included in the mutation assay with metabolic activation (+S9) for the same reason and also to demonstrate the efficacy of the S9-cofactor mix. In addition, since CPA is known to cause chromosomal damage (resulting in small colonies) as well as to point mutations, its use should demonstrate the sensitivity of the test system to clastogens.
In all cases, duplicate cultures were treated with a single positive control concentration in dimethyl sulphoxide (DMSO) at a volume of 1% of the exposure medium. The use of DMSO at this level has been shown previously to be acceptable and is customarily used in this test system.
The mutant frequency (MF) for each treatment was calculated:
MF for each culture was calculated from the proportion of wells without colonies in selective medium plates, P(0)MF. It was corrected for viability using CE (above) calculated from the proportion of wells without colonies in nonselective medium, P(0)CE. Where the selective medium plates were seeded with 2000 cells per well, and the non-selective medium plates with 1.6 cells per well:
MF P (0) = (Total number of wells – number of wells with a colony) / Total number of wells
Individual MF = (−ln P(0) Mutant Plates / 2000 x (Viability (CE)) / 100)) x 1000,000
Average MF (pooled) = (MF total for that treatment condition / Total number of replicates for that treatment condition)
Induced Mutant Frequency (IMF) calculations:
Pooled IMF = Mean MF of replicate culture (test substance) - Mean MF of control
Pooled IMF small = Mean small MF of replicate culture (test substance) - Mean small IMF of control
IMF small colonies % = (IMF (small) of replicate culture (test substance) / IMF of pooled cultures (test substance)) x100 - Evaluation criteria:
- I] Criteria for conclusion of a clearly positive result:
The criteria for concluding that an individual test is clearly positive was:
a) the MF at one or more concentrations is at least 126 x 10˄-6 greater than the concurrent solvent control (GEF) and
b) there is a significant dose-relationship as indicated by trend test analysis and
c) any positive results obtained at less than 10% RTG are excluded
The positive control cultures mutant frequency values should approximate those of the acceptable ranges from the Test Facility’s historical control database.
II] Criteria for conclusion of a clearly negative result:
The criterion for concluding that an individual test is negative (considered non-mutagenic) will be:
a) The GEF is not exceeded at any test substance concentration.
b) There is no concentration related increase. A trend test will be conducted if the GEF is exceeded or if the Study Director feels that it may aid in the interpretation of a possible concentration related increase that may be just below the GEF. - Statistics:
- Please see 'Any other information on materials and methods incl. tables' for information on Statistics.'
- Key result
- Species / strain:
- mouse lymphoma L5178Y cells
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- True negative controls validity:
- not specified
- Positive controls validity:
- valid
- Additional information on results:
- TEST-SPECIFIC CONFOUNDING FACTORS
There were no remarkable changes in osmolality or pH observed between the solvent controls and test concentrations evaluated using an osmometer and pH meter, respectively.
RANGE-FINDING/SCREENING STUDIES:
Treatment schedule: 3 hours in the absence of metabolic activation (-S9)
The concentration range tested was 21.62 to 1500 μg/mL. Upon addition of test material to the culture medium, precipitate was observed at a concentration of 491.5 μg/mL and above. At the end of the treatment period, precipitate was observed at a concentration of 1500 μg/mL and above. Cytotoxicity (identified as a reduction in RSG of less than 10%) was observed at a concentration of 314.6 μg/mL and above. Lower concentrations of 251.7, 201.3, 161.1, 128.8, 103.1, 82.46, 65.97, 52.78, 42.22, 33.78, 27.02 and 21.62 μg/mL yielded mean RSG values of 28, 78, 94, 94, 94, 100, 100, 100, 94, 100, 94, and 94%, respectively.
Treatment schedule: 3 hours in the presence of metabolic activation (+S9)
The concentration range tested was 21.62 to 1500 μg/mL. Upon addition of test material to the culture medium, precipitate was observed at a concentration of 491.5 μg/mL and above. At the end of the treatment period, precipitate was observed at a concentration of 1500 μg/mL. Cytotoxicity (identified as a reduction in RSG of less than 10%) was observed at a concentration of 1500 μg/mL. Lower concentrations of 1200, 960.0, 768.0, 614.4, 491.5, 393.2, 314.6, 251.7, 201.3, 161.1, 128.8, 103.1, 82.46, 65.97,52.78, 42.22, 33.78, 27.02 and 21.62 μg/mL yielded mean RSG values of 11, 32, 47, 63, 68, 63, 58, 68, 63, 68, 74, 79, 84, 79, 79, 84, 89, 89, and 95%, respectively.
STUDY RESULTS
- Concurrent vehicle negative and positive control data:
The mean solvent control (DMSO) suspension growth, cloning efficiency, and mutant frequency were consistent with those stated in the OECD guideline (490) and current historical control data. The results obtained with positive control agents demonstrated the ability of the test system to identify mutagens and clastogens and where appropriate, the ability of the S9 mix to metabolise cyclophosphamide.
Main Mutagenicity Experiments:
Experiment 1: Treatment schedule: 3 hours in the absence of metabolic activation (-S9):
The concentration range tested was 37.62 to 314.6 μg/mL. Upon addition of test item to the culture medium, precipitate was observed at a concentration of 229.3 μg/mL and above. At the end of the treatment period, no precipitate was observed at any concentration tested.
A mean RSG value of 12% was observed at a concentration of 314.6 μg/mL. Lower concentrations of 283.1, 254.8, 229.3, 206.4, 185.8, 167.2, 150.5, 75.24and 37.62 μg/mL yielded mean RSG values of 1, 25, 17, 16, 3, 6, 12, 103, and 102%, respectively. Due to variability and lack of coverage in the RSG values, if plated it was unlikely that the RTG values would have covered the cytotoxicity range, from that producing cytotoxicity and including concentrations at which there is moderate and little or no cytotoxicity. Therefore, the cultures were discarded and further testing was conducted.
Experiment 2: Treatment schedule: 3 hours without metabolic activation (-S9):
The concentration range tested was 39.98 to 314.6 μg/mL. Upon addition of test item to the culture medium, precipitate was observed at a concentration of 206.4 μg/mL and above. At the end of the treatment period, no precipitate was observed at any concentration tested.
A mean RSG value of 1% was observed at a concentration of 314.6 μg/mL. Lower concentrations of 283.1, 254.8, 229.3, 206.4, 185.8, 167.2, 150.5, 135.4, 121.9, 109.7, 98.72, 88.85, 79.97 and 39.98 μg/mL yielded mean RSG values of 1, 2, 3, 3, 5, 8, 10, 17, 32, 41. 65. 67. 78 and 97%, respectively. The following concentrations were selected for plating 150.5, 135.4, 121.9, 109.7, 98.72, 79.97, and 39.98 μg/mL. A mean RTG value of 8% (cytotoxicity of 92%) was observed at a concentration of 150.5 μg/mL. Lower concentrations of 135.4, 121.9, 109.7, 98.72, 79.97, and 39.98 μg/mL yielded mean RTG values of 11, 25, 32, 57, 78, and 101%, respectively.
Data for the SG, CE, and MF in the solvent control of the 3 hour treatment without S9 mix were consistent with those stated in the OECD guideline and the MF was consistent with the test facility’s historical control database (based on 95% confidence limits) for L5178Y cells and therefore, the acceptance criteria were met. Data for the small colony IMF (174 x 10˄-6) in the 4NQO positive control result of the 3 hour treatment without S9 mix were consistent with the acceptance criteria stated in the OECD guidelineand the test facility’s historical control database and a mean RTG value of 87% was observed. The mutant frequencies of the test item did not exceed the GEF at any concentrations analysed. As no concentration came close to meeting or exceeding the GEF a trend-test was not conducted as there was no apparent evidence of a relevant concentration related increase in mutant frequency. The criteria for a clear negative result were met.
Experiment 1: Treatment schedule: 3 hours with metabolic activation (+S9):
The concentration range tested was 19.20 to 1500 μg/mL. Upon addition of test item to the culture medium, precipitate was observed at a concentration of 1080 µg/mL and above. At the end of the treatment period, no precipitate was observed at any concentration tested.
A mean RSG value of 76% was observed at a concentration of 1500 μg/mL. Lower concentrations of 1200, 1080, 960, 768.0, 614.4, 307.2, 153.6, 76.80, 38.40, and 19.20 μg/mL yielded mean RSG values of 11, 46, 58, 78, 80, 84, 89, 86, 93, and 97%, respectively. Due to the lack of cytotoxicity observed at the top dose (1500 µg/mL), leading to inconsistencies in RSG values and the dose response, the cultures were discarded and further testing was conducted.
Experiment 2: Treatment schedule: 3 hours with metabolic activation (+S9):
The concentration range tested was 19.20 to 1500 μg/mL. Upon addition of test item to the culture medium, precipitate was observed at a concentration of 614.4 µg/mL and above. At the end of the treatment period, no precipitate was observed at any concentration tested.
A mean RSG value of 2% was observed at a concentration of 1500 μg/mL. Lower concentrations of 1200, 1140, 1083, 960.0, 768.0, 614.4, 307.2, 153.6, 76.80, 38.40, and 19.20 μg/mL yielded mean RSG values of 3, 5, 8, 14, 33, 56, 68, 70, 81, 89, and 89%, respectively. The following concentrations were selected for plating 1200, 960.0, 768.0, 614.4, 307.2 and 38.40 μg/mL.
A mean RTG of 1% (cytotoxicity of 99%) was observed at a concentration of 1200 μg/mL. Lower concentrations of 960.0, 768.0, 614.4, 307.2, and 38.40 μg/mL gave RTG values of 10, 26, 54, 64, and 101%, respectively. However, the concentration 1200 µg/mL was excluded as the RTG was below 10%, in accordance with the OECD guideline. Furthermore, a high CE value for a 38.40 µg/mL culture was recorded, this was thought to be due to a sampling error when preparing the cell suspension for plating. Therefore, this culture was excluded from the data analysis; this is deemed as acceptable as single cultures can be dosed in accordance with the OECD guidelines and will not impact the validity and integrity of the study.
Data for the SG, CE and MF in the solvent control of the 3 hour treatment with S9 mix were consistent those stated in the OECD guideline and the MF was consistent with the test facility’s historical control database (based on 95% confidence limits) for L5178Y cells and therefore, the acceptance criteria were met. Data for the small colony IMF (507x10-6) in the CPA positive control result of the 3 hour with S9 mix were consistent with the acceptance criteria stated in the OECD guideline. The CPA mutant frequency (1416x10-6) was higher than the upper 95% confidence limits of the historical positive control (165-1342 x10˄-6), however this was considered acceptable since it demonstrated a positive response could be induced. A mean RTG value of 52% was observed for the CPA. The mutant frequencies of the test item did not exceed the GEF at any concentrations analysed. As no concentration came close to meeting or exceeding the GEF a trend-test was not conducted as there was no apparent evidence of a relevant concentration related increase in mutant frequency The criteria for a clear negative result were met.
There was no indication of a requirement to conduct testing for 24 hours without S9, as outlined in the OECD guideline. Namely, the test item was readily soluble and is not known to be of a class of substances that require longer exposure times to be detected (e.g. a nucleoside analogue). Further testing with a 24 hours without S9 treatment schedule was not required for this test item, this has no impact on the validity and integrity of this study.
HISTORICAL CONTROL DATA (with ranges, means and standard deviation, and 95% control limits for the distribution as well as the number of data)
- Positive historical control data: Please see 'Table 4' in 'Any other information on results incl. tables' for information on historical control data.
- Negative (solvent/vehicle) historical control data: Please see 'Table 4' in 'Any other information on results incl. tables' for information on historical control data. - Conclusions:
- Petroleum Gas Oil fraction, Co-processed with Renewable Hydrocarbons of Plant and/or Animal origin (EC 941-364-9) was not mutagenic in the mouse lymphoma L5178Y Tk locus assay in the absence and presence of S9 mix, and under the test conditions used.
- Executive summary:
In a key in vitro mammalian cell gene mutation assay, the potential of the test material (Petroleum Gas Oil fraction, Co-processed with Renewable Hydrocarbons of Plant and/or Animal origin (EC 941-364-9)) to induce forward mutation at the thymidine kinase (Tk) locus in mouse lymphoma L5178Y TK+/-3.7.2 C cells was evaluated. Treatment concentrations for the mutation assays were selected on the basis of the result of a range-finding preliminary cytotoxicity test.
The test material was applied to the test system under two treatment schedules: treatment for 3 hours in the absence and presence of an in vitro activation system based on S9 fraction obtained from Aroclor 1254-induced rat liver (S9 mix). Dimethyl sulphoxide (DMSO) was used as vehicle in this study.
The following test material concentrations were examined in the mutation assays:
Range finding experiment:
21.62, 29.02, 33.78, 42.22, 52.78, 65.97, 82.46, 103.1, 128.8, 161.1, 201.3, 251.7, 314.6, 393.2, 491.5, 614.4, 768.0, 960.0, 1200, 1500 μg/mL.
Main Mutation Experiment 01 (3-hour treatment):
(-S9): 39.98 to 314.6 μg/mL
(+S9): 19.20 to 1200 μg/mL
Data for the suspension growth (SG), CE and Mutant Frequency (MF) in the solvent control for all treatment schedules were consistent with those stated in the OECD guideline and the MF was consistent with the test facility’s historical control database (based on 95% confidence limits) for L5178Y cells and therefore, the acceptance criteria were met. The positive control results for all treatment schedules were consistent with the acceptance criteria stated in the OECD guideline.
In Experiment 02, 3-hour -S9 treatment exposure: a concentration of 150.5 µg/mL gave 8% relative total growth (RTG) (cytotoxicity of 92%). In Experiment 02, 3-hour +S9 treatment exposure: a concentration of 960.0 µg/mL gave 10% RTG (cytotoxicity of 90%).
In final mutation assay experiments, upon addition of the test item to the culture medium, precipitate was observed at a concentration of 206.4 µg/mL and above in the 3h –S9 mix treatment schedule and at a concentration of 614.4 µg/mL and above in the 3h +S9 mix treatment schedule. At the end of the treatment period, no precipitate was observed in either treatment schedule.
No relevant changes in mutant frequency (MF) were observed for any of the treatment schedules at any of the concentrations analysed. The Global Evaluation Factor (GEF) was not exceeded for any of the test concentrations analysed and the criteria for a clear negative response were met for all treatment schedules.
Under the test conditions used, Petroleum Gas Oil fraction, Co-processed with Renewable Hydrocarbons of Plant and/or Animal origin (EC 941-364-9) was not mutagenic in the mouse lymphoma L5178Y Tk locus assay in the absence and presence of S9 mix.
- Endpoint:
- in vitro cytogenicity / micronucleus study
- Type of information:
- experimental study
- Adequacy of study:
- supporting study
- Study period:
- 18-Jan-2021 to 25-Mar-2021
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)
- GLP compliance:
- yes (incl. QA statement)
- Type of assay:
- in vitro mammalian cell micronucleus test
- Species / strain / cell type:
- human lymphoblastoid cells (TK6)
- Metabolic activation:
- with and without
- Metabolic activation system:
- Aroclor 1254 induced S9
- Test concentrations with justification for top dose:
- The final concentration ranges tested from which micronuclei were analysed for were:
3h +S9 treatment schedule: 296.3 to 1500 µg/mL
3h -S9 treatment schedule : 666.7 to 1500 µg/mL
continuous treatment schedule (24h -S9): 131.7 to 666.7 µg/mL
The following levels of cytotoxicity were observed, recommended maximum level of cytotoxicity (55 ± 5%) was observed in the continuous and 3h -S9 treatment schedules:
3h +S9 treatment schedule: 42.67% at 1500 µg/mL
3h -S9 treatment schedule: 52.84% at 1500 µg/mL
continuous treatment schedule (24h -S9): 55.22% at 666.7 µg/mL
For a test item exhibiting apparent cytotoxicity, the aim was that the highest concentration selected was that which yielded cytotoxicity of ~55 ± 5%. Further doses were selected from those yielding decreasing levels of cytotoxicity, as far as a no-effect dose (little or no cytotoxicity by CBPI).
For test items that did not yield apparent cytotoxicity, the three highest test concentrations were selected for further microscopic analysis. - Vehicle / solvent:
- Dimethyl sulfoxide (DMSO)
- Negative solvent / vehicle controls:
- yes
- Positive controls:
- yes
- Positive control substance:
- colchicine
- cyclophosphamide
- mitomycin C
- Details on test system and experimental conditions:
- Test system
The study was designed to meet the requirements of OECD Guideline 487 (2016) and was agreed with the sponsor prior to commencement of the study. Since this study utilised isolated human lymphocytes and hence used cytokinesis block protocols, the specific guidance (in OECD 487) relating to lymphocytes and studies performed in the presence of cytoB, was followed.
Experimental conditions
Human lymphocytes were isolated from pooled anti-coagulated fresh blood obtained from two healthy non-smoking males and allowed to proliferate for approximately 44 to 48 hours in the presence of the mitogen, phytohaemagglutinin (PHA). Cultures of proliferating lymphocytes were exposed to the test item in the presence of S9 mix for 3 hours before cells were washed and then further incubated (humidified atmosphere of 5% CO2 at a temperature of 37oC) in fresh medium containing cytoB for approximately 21 hours. Treatments in the absence of S9 mix were performed for a 3-hour period (followed by cell washing and incubation in fresh medium containing cytoB for approximately 21 hours) and for a continuous exposure period for 24 hours. In the continuous treatment condition, cytoB was added concurrently with the test item. Duplicate cultures were treated for each test item concentration and positive control. For the solvent-treated controls, four cultures were dosed with DMSO. - Evaluation criteria:
- Criteria for a clearly positive response:
- at least one of the test concentrations exhibited a statistically significant increase compared with the concurrent negative control
- the increase was dose-related in at least one experimental condition when evaluated with an appropriate trend test
- any of the results were outside the distribution of the historical solvent control range (Poisson-based 95% control limits)
Criteria for a clearly negative response
- none of the test concentrations exhibited a statistically significant increase compared with the concurrent negative control
- there was no concentration-related increase when evaluated using an appropriate trend test
- all results were inside the distribution of the historical negative control data (Poisson-based 95% control limits)
Criteria for an equivocal response
Although most experiments are expected to yield clearly positive or negative results, in some cases the data preclude making a definitive judgement about the activity of the test item. Such equivocal responses may occur regardless of the number of times the experiment was repeated. - Statistics:
- The number of micronuclei analysed from 2000 binucleated cells for each selected test item dose was compared with that from the concurrent solvent control. Pair-wise statistical analysis employing a one-sided Fisher’s Exact test were used to evaluate statistical significance (p < 0.05). A linear trend test was employed (Cochran-Armitage) in order to confirm there was no dose related increase (p < 0.05).
- Key result
- Species / strain:
- lymphocytes: micronuclei
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity, but tested up to precipitating concentrations
- Vehicle controls validity:
- valid
- Positive controls validity:
- valid
- Remarks on result:
- other: all strains/cell types tested
- Remarks:
- no mutagenic potential
- Conclusions:
- It was concluded that Petroleum Gas Oil Fraction, Co-processed with Renewable Hydrocarbons of Plant and/or Animal Origin (EC 941-364-9) did not induce the formation of micronuclei (MN) in human lymphocytes in the absence and presence of S9, under the test conditions used. The criteria for a negative response were met.
- Executive summary:
Petroleum Gas Oil Fraction, Co-processed with Renewable Hydrocarbons of Plant and/or Animal Origin (EC 941-364-9) was tested for its potential to induce micronucleus formation in thein vitro micronucleus test with manual scoring on microscope slides. In all tests none of the treatment schedules resulted in significant increases in micronucleus formation in test item treated cultures. It was concluded that Petroleum Gas Oil Fraction, Co-processed with Renewable Hydrocarbons of Plant and/or Animal Origin (EC 941-364-9) did not induce the formation of micronuclei (MN) in human lymphocytes in the absence and presence of S9, under the test conditions used. The criteria for a negative response were met.
Referenceopen allclose all
Table 1 – Test 1: Petroleum Disel/Gas Oil Fraction, Co-processed with Renewable Hydrocarbons Derived from Thermally Cracked Plastics (EC# 941-803-4): plate incorporation method – with metabolic activation
Dose per plate |
Number of revertant colonies per plate |
|||||||||
S. typhimuriumLT2 |
E. coliWP2 |
|||||||||
|
TA1535 |
TA1537 |
TA98 |
TA100 |
uvrA/pKM101 |
|||||
μg |
Mean |
SD |
Mean |
SD |
Mean |
SD |
Mean |
SD |
Mean |
SD |
Solvent control |
12 |
1.5 |
10 |
0.6 |
33 |
1.7 |
144 |
11.0 |
196 |
3.2 |
1.6 |
13 |
2.3 |
8 |
1.5 |
31 |
1.2 |
143 |
17.1 |
195 |
16.2 |
5 |
11 |
1.5 |
9 |
0.6 |
30 |
3.5 |
153 |
9.3 |
205 |
3.2 |
16 |
13 |
1.5 |
11 |
3.0 |
33 |
3.8 |
139 |
15.6 |
204 |
4.0 |
50 |
12 |
0.0 |
11 |
1.5 |
32 |
2.6 |
131 |
11.0 |
208 |
3.5 |
160 |
12 |
0.6 |
10 |
2.1 |
36 |
3.0 |
144 |
7.1 |
190 |
4.2 |
500 |
11 |
1.2 |
10 |
0.6 |
39 |
1.7 |
134 |
6.1 |
183 |
4.6 |
1600 |
11 |
1.2 |
11 |
0.6 |
32 |
1.7 |
146 |
15.9 |
195 |
8.1 |
5000 |
10 |
1.5 |
12 |
1.5 |
38 |
3.8 |
152 |
12.0 |
194 |
3.6 |
Positive control |
162 |
6.8 |
148 |
26.1 |
1370 |
312.6 |
1772 |
336.5 |
1883 |
172.0 |
Positive controls
TA1535: 2-aminoanthracene 2 μg/plate
TA1537: 2-aminoanthracene 2 μg/plate
TA98: 2-aminoanthracene 2 μg/plate
TA100: 2-aminoanthracene 2 μg/plate
uvrA/pKM101: 2-aminoanthracene 20 μg/plate
Table 2 – Test 1: Petroleum Disel/Gas Oil Fraction, Co-processed with Renewable Hydrocarbons Derived from Thermally Cracked Plastics (EC# 941-803-4): plate incorporation method – without metabolic activation
Dose per plate |
Number of revertant colonies per plate |
|||||||||
S. typhimuriumLT2 |
E. coliWP2 |
|||||||||
|
TA1535 |
TA1537 |
TA98 |
TA100 |
uvrA/pKM101 |
|||||
μg |
Mean |
SD |
Mean |
SD |
Mean |
SD |
Mean |
SD |
Mean |
SD |
Solvent control |
13 |
1.2 |
12 |
0.6 |
27 |
0.6 |
122 |
6.8 |
171 |
6.4 |
1.6 |
12 |
1.0 |
11 |
1.5 |
26 |
1.2 |
113 |
4.6 |
169 |
1.5 |
5 |
13 |
1.0 |
11 |
0.0 |
31 |
3.1 |
119 |
4.2 |
164 |
9.5 |
16 |
12 |
1.5 |
10 |
1.0 |
28 |
2.1 |
114 |
3.5 |
160 |
20.4 |
50 |
13 |
1.0 |
11 |
0.6 |
28 |
2.6 |
118 |
4.0 |
165 |
6.1 |
160 |
12 |
0.6 |
10 |
1.0 |
24 |
2.0 |
123 |
6.2 |
154 |
6.0 |
500 |
12 |
2.0 |
11 |
1.5 |
31 |
0.6 |
119 |
4.4 |
155 |
12.1 |
1600 |
13 |
0.6 |
9 |
1.2 |
27 |
4.4 |
119 |
9.9 |
147 |
3.1 |
5000 |
10 |
2.5 |
6 |
1.7 |
27 |
3.8 |
110 |
6.6 |
147 |
6.1 |
Positive control |
427 |
26.4 |
216 |
25.2 |
234 |
62.4 |
472 |
8.5 |
988 |
46.8 |
Positive controls
TA1535: sodium azide
0.5 μg/plate
TA1537: 2-aminocridine.HCl 50 μg/plate
TA98: 2-nitrofluorene 1 μg/plate
TA100: sodium azide 0.5 μg/plate
uvrA/pKM101: potassium dichromate 25 μg/plate
Table 3 – Test 2: Petroleum Disel/Gas Oil Fraction, Co-processed with Renewable Hydrocarbons Derived from Thermally Cracked Plastics (EC# 941-803-4): liquid pre-incubation method – with metabolic activation
Dose per plate |
Number of revertant colonies per plate |
|||||||||
S. typhimuriumLT2 |
E. coliWP2 |
|||||||||
|
TA1535 |
TA1537 |
TA98 |
TA100 |
uvrA/pKM101 |
|||||
μg |
Mean |
SD |
Mean |
SD |
Mean |
SD |
Mean |
SD |
Mean |
SD |
Solvent control |
11 |
1.7 |
12 |
1.5 |
36 |
4.5 |
134 |
5.3 |
197 |
8.3 |
1.6 |
14 |
1.5 |
10 |
1.7 |
32 |
6.7 |
146 |
7.0 |
200 |
17.0 |
5 |
13 |
1.2 |
10 |
1.0 |
31 |
6.4 |
148 |
8.5 |
202 |
15.4 |
16 |
11 |
1.2 |
11 |
1.2 |
33 |
6.1 |
148 |
6.1 |
191 |
14.2 |
50 |
9 |
2.1 |
11 |
1.2 |
33 |
2.3 |
133 |
10.8 |
208 |
9.0 |
160 |
10 |
0.6 |
10 |
0.6 |
31 |
10.4 |
130 |
2.5 |
192 |
14.7 |
500 |
10 |
1.0 |
12 |
1.5 |
41 |
1.7 |
150 |
9.3 |
194 |
20.1 |
1600 |
11 |
2.1 |
10 |
1.5 |
34 |
4.2 |
145 |
24.2 |
190 |
18.6 |
5000 |
9 |
0.6 |
11 |
1.2 |
39 |
2.6 |
119 |
4.4 |
165 |
15.5 |
Positive control |
189 |
9.5 |
205 |
18.8 |
2149 |
163.2 |
3093 |
20.6 |
1947 |
81.8 |
Positive controls
TA1535:
2-aminoanthracene 2 μg/plate
TA1537: 2-aminoanthracene 2 μg/plate
TA98: 2-aminoanthracene 2 μg/plate
TA100: 2-aminoanthracene 2 μg/plate
uvrA/pKM101: 2-aminoanthracene 20 μg/plate
Table 4 – Test 2: Petroleum Disel/Gas Oil Fraction, Co-processed with Renewable Hydrocarbons Derived from Thermally Cracked Plastics (EC# 941-803-4): liquid pre-incorporation method – without metabolic activation
Dose per plate |
Number of revertant colonies per plate |
|||||||||
S. typhimuriumLT2 |
E. coliWP2 |
|||||||||
|
TA1535 |
TA1537 |
TA98 |
TA100 |
uvrA/pKM101 |
|||||
μg |
Mean |
SD |
Mean |
SD |
Mean |
SD |
Mean |
SD |
Mean |
SD |
Solvent control |
13 |
1.2 |
10 |
0.0 |
31 |
2.6 |
119 |
2.1 |
159 |
9.0 |
1.6 |
12 |
1.2 |
9 |
1.2 |
31 |
1.2 |
110 |
5.2 |
148 |
10.1 |
5 |
12 |
1.0 |
9 |
0.6 |
29 |
4.6 |
117 |
2.6 |
169 |
6.5 |
16 |
12 |
0.6 |
10 |
2.1 |
28 |
5.1 |
116 |
3.8 |
162 |
15.7 |
50 |
13 |
1.2 |
8 |
1.5 |
30 |
4.5 |
115 |
3.1 |
147 |
14.4 |
160 |
11 |
1.2 |
10 |
0.6 |
26 |
3.2 |
111 |
0.6 |
146 |
8.2 |
5000 |
12 |
1.7 |
9 |
0.6 |
29 |
2.5 |
116 |
4.5 |
135 |
2.5 |
1600 |
12 |
1.5 |
6 |
0.6 |
26 |
3.6 |
112 |
4.0 |
132 |
7.4 |
5000 |
7 |
2.0 |
4 |
1.7 |
23 |
5.3 |
83 |
4.2 |
88 |
1.6 |
Positive control |
415 |
50.3 |
306 |
121.4 |
812 |
64.8 |
523 |
82.8 |
958 |
9.0 |
Positive controls
TA1535: sodium azide
0.5 μg/plate
TA1537: 2-aminocridine.HCl 50 μg/plate
TA98: 2-nitrofluorene 1 μg/plate
TA100: sodium azide 0.5 μg/plate
uvrA/pKM101: potassium dichromate 25 μg/plate
Micronucleus data
Table 3 – Petroleum Diesel/Gas Oil Fraction, Co-processed with Renewable Hydrocarbons derived from thermally cracked plastics (EC 941 -803 -4): 3 hour exposure testing in the presence of S9 mix (Experiment 01)
Dose (µg/mL) | 3h + S9 treatment | |||
CBPI | % Cytostasis | % MN per dose | p-value | |
Solvent (DMSO) | 1.540 | N/A | 1.0 | N/A |
1481 | 1.530 | 1.91 | 1.1 | ns |
2222 | 1.380 | 29.61 | 1.1 | ns |
3333P | 1.229 | 57.58 | 1.0 | ns |
CPA 4 | 1.401 | 25.68 | 3.4 | 4.553 x 10-08 |
CBPI = cytokinesis block proliferation index
MN per dose = micronucleated binucleates/2000 binucleates
CPA = cyclophosphamide
P = precipitate observed at the end of the treatment period
ns = not statistically significant (by Fisher’s exact test), % - individual percentages of micronucleated binucleated cells,Cochran-Armitage treated test p-value = 8.445×10-1, therefore not statistically significant
Table 4- Petroleum Diesel/Gas Oil Fraction, Co-processed with Renewable Hydrocarbons derived from thermally cracked plastics (EC 941 -803 -4): 3 hour exposure testing in the absence of S9 mix (Experiment 01)
Dose (µg/mL) | 3h - S9 treatment | |||
CBPI | % Cytostasis | % MN per dose | p-value | |
Solvent (DMSO) | 1.496 | N/A | 1.1 | N/A |
1481 | 1.609 | -22.73 | 1.1 | ns |
2222 | 1.416 | 16.13 | 1.0 | ns |
3333P | 1.304 | 38.71 | 0.9 | ns |
MMC 50 ng/mL | 1.390 | 21.38 | 3.9 | 3.653 x10-09 |
CBPI = cytokinesis block proliferation index
MN per dose = micronucleated binucleates/2000 binucleates
MMC = mitomycin
P = precipitate observed at the end of the treatment period
ns = not statistically significant (by Fisher’s exact test), % - individual percentages of micronucleated binucleated cells,Cochran-Armitage treated test p-value = 5.309×10-1, therefore not statistically significant
Table 5- Petroleum Diesel/Gas Oil Fraction, Co-processed with Renewable Hydrocarbons derived from thermally cracked plastics (EC 941 -803 -4): continuous (24 hour) exposure testing in the absence of S9 mix (Experiment 01)
Dose (µg/mL) | 24h - S9 treatment | |||
CBPI | % Cytostasis | % MN per dose | p-value | |
Solvent (DMSO) | 1.359 | N/A | 1.0 | N/A |
292.6 | 1.530 | -47.52 | 1.0 | ns |
439.0 | 1.252 | 29.86 | 1.0 | ns |
658.4 | 1.242 | 32.64 | 1.0 | ns |
987.7 | 1.72 | 52.13 | 1.1 | ns |
MMC 30 ng/mL | 1.314 | 12.60 | 3.3 | 1.14 x 10-07 |
COL 7.5 ng/mL | 1.398 | -10.78 | 2.8 | 9.426 x 10-06 |
CBPI = cytokinesis block proliferation index
MN per dose = micronucleated binucleates/2000 binucleates
MMC = mitomycin
COL = colchicine
P = precipitate observed at the end of the treatment period
ns = not statistically significant (by Fisher’s exact test), % - individual percentages of micronucleated binucleated cells,Cochran-Armitage treated test p-value = 8.295×10-01, therefore not statistically significant
Cytotoxicity assessment
Table 6- Petroleum Diesel/Gas Oil Fraction, Co-processed with Renewable Hydrocarbons derived from thermally cracked plastics (EC 941 -803 -4): cytotoxicity assessment in cytokinesis blocked lymphocytes from 3 hour exposure testing in the presence of S9 mix (Experiemnt 01)
Concentration (µg/mL) | Number of nucleated cells | CBPI | % Cytostasis | |||
Mono | Bi | Multi | Total scored | |||
Solvent (DMSO) | 245 | 243 | 14 | 502 | 1.540 | 0.00 |
17.13 | sns | sns | sns | sns | ND | ND |
25.69 | sns | sns | sns | sns | ND | ND |
38.54 | sns | sns | sns | sns | ND | ND |
57.81 | sns | sns | sns | sns | ND | ND |
86.71 | sns | sns | sns | sns | ND | ND |
130.1 | sns | sns | sns | sns | ND | ND |
195.1 | sns | sns | sns | sns | ND | ND |
292.6 | sns | sns | sns | sns | ND | ND |
439.0 | sns | sns | sns | sns | ND | ND |
658.4 | sns | sns | sns | sns | ND | ND |
987.7 | 260 | 236 | 5 | 501 | 1.491 | 9.04 |
1481 | 243 | 261 | 4 | 508 | 1.530 | 1.91 |
2222 | 315 | 180 | 5 | 500 | 1.380 | 29.61 |
3333 P | 404 | 120 | 0 | 524 | 1.229 | 57.58 |
5000 P | snp | snp | snp | snp | ND | ND |
CPA 4 µg/mL | 300 | 201 | 0 | 501 | 1.401 | 25.68 |
Mono = mononucleated cells
Bi = binucleated cells
Multi = multinucleated cells
total scored = sum of mono-, bi- and multi-
CBPI = cytokinesis block proliferation index
CPA = cyclophosphamide
sns = slide not scored/analysed
ND = not determined
P = precipitate observed at the end of the treatment period
Table 7 -Petroleum Diesel/Gas Oil Fraction, Co-processed with Renewable Hydrocarbons derived from thermally cracked plastics (EC 941 -803 -4):cytotoxicity assessment in cytokinesis blocked lymphocytes from 3 hour exposure testing in the absence of S9 mix (Experiment 01)
Concentration (µg/mL) | Number of nucleated cells | CBPI | % Cytostasis | |||
Mono | Bi | Multi | Total scored | |||
Solvent (DMSO) | 256 | 246 | 2 | 504 | 1.496 | 0.00 |
17.13 | sns | sns | sns | sns | ND | ND |
25.69 | sns | sns | sns | sns | ND | ND |
38.54 | sns | sns | sns | sns | ND | ND |
57.81 | sns | sns | sns | sns | ND | ND |
86.71 | sns | sns | sns | sns | ND | ND |
130.1 | sns | sns | sns | sns | ND | ND |
195.1 | sns | sns | sns | sns | ND | ND |
292.6 | sns | sns | sns | sns | ND | ND |
439.0 | sns | sns | sns | sns | ND | ND |
658.4 | sns | sns | sns | sns | ND | ND |
987.7 | 237 | 260 | 4 | 501 | 1.535 | -7.84 |
1481 | 199 | 299 | 3 | 501 | 1.609 | -22.73 |
2222 | 293 | 206 | 1 | 500 | 1.416 | 16.13 |
3333 P | 348 | 152 | 0 | 500 | 1.304 | 38.17 |
5000 P | snp | snp | snp | snp | ND | ND |
MMC 50 ng/mL | 305 | 195 | 0 | 500 | 1.390 | 21.38 |
Mono = mononucleated cells
Bi = binucleated cells
Multi = multinucleated cells
total scored = sum of mono-, bi- and multi-
CBPI = cytokinesis block proliferation index
MMC = mitomycin C
sns = slide not scored/analysed
ND = not determined
P = precipitate observed at the end of the treatment period
Table 8 -Petroleum Diesel/Gas Oil Fraction, Co-processed with Renewable Hydrocarbons derived from thermally cracked plastics (EC 941 -803 -4):cytotoxicity assessment in cytokinesis blocked lymphocytes from continuous (24 hour) exposure testing in the absence of S9 mix (Experiment 01)
Concentration (µg/mL) | Number of nucleated cells | CBPI | % Cytostasis | |||
Mono | Bi | Multi | Total scored | |||
Solvent (DMSO) | 325 | 172 | 4 | 501 | 1.359 | 0.00 |
17.13 | sns | sns | sns | sns | ND | ND |
25.69 | sns | sns | sns | sns | ND | ND |
38.54 | sns | sns | sns | sns | ND | ND |
57.81 | sns | sns | sns | sns | ND | ND |
86.71 | sns | sns | sns | sns | ND | ND |
130.1 | sns | sns | sns | sns | ND | ND |
195.1 | 242 | 253 | 5 | 500 | 1.526 | -46.40 |
292.6 | 239 | 257 | 4 | 500 | 1.530 | -47.52 |
439.0 | 376 | 122 | 2 | 500 | 1.252 | 29.86 |
658.4 | 383 | 113 | 4 | 500 | 1.242 | 32.64 |
987.7 | 417 | 80 | 3 | 500 | 1.172 | 52.13 |
1481 | 502 | 58 | 0 | 560 | 1.104 | 71.17 |
2222 P | tfbts | tfbts | tfbts | tfbts | ND | ND |
3333 P | snp | snp | snp | snp | ND | ND |
5000 P | snp | snp | snp | snp | ND | ND |
MMC 30 ng/mL | 344 | 155 | 1 | 500 | 1.314 | 12.60 |
Col 7.5 ng/mL | 312 | 177 | 11 | 500 | 1.398 | -10.78 |
Mono = mononucleated cells
Bi = binucleated cells
Multi = multinucleated cells
total scored = sum of mono-, bi- and multi-
CBPI = cytokinesis block proliferation index
MMC = mitocyin C
sns = slide not scored/analysed
ND = not determined
P = precipitate observed at the end of the treatment period
tfbts = too few binucleates to score
Table 2. Summary of Analysed Concentrations | |||||||||
Exposure | Test | RTG | SG | CE | MF ( x10-6)* | IMF | IMF | IMF | (MF) |
3 hour -S9 mix | Solvent | 100 | 19 | 112 | 86 | 0 | 0 | 0 | 58 to 119 |
GEF threshold | N/A | N/A | N/A | 212 | N/A | N/A | N/A | N/A | |
85.71 | 90 | 19 | 98 | 103 | 17 | 2 | |||
107.1 | 82 | 17 | 104 | 102 | 16 | 3 | |||
133.9 | 49 | 10 | 103 | 95 | 8 | 6 | |||
167.4 | 22 | 4 | 109 | 134 | 47 | 17 | |||
186.0 | 12 | 2 | 101 | 120 | 34 | 8 | |||
206.7 | 7 | 2 | 97 | 139 | 53 | 15 | |||
4NQO 0.19 | 58 | 15 | 85 | 1073 | 987 | 314 | 32 | 431 to 1177 | |
3 hour +S9 mix | Solvent | 100 | 23 | 114 | 75 | 0 | 0 | 0 | 55 to 123 |
GEF threshold | N/A | N/A | N/A | 201 | N/A | N/A | N/A | N/A | |
43.11 | 88 | 22 | 103 | 107 | 31 | 20 | |||
172.4 | 69 | 17 | 103 | 125 | 49 | 13 | |||
344.9 | 57 | 11 | 129 | 77 | 2 | 5 | |||
862.2 | 34 | 9 | 102 | 155 | 79 | 30 | |||
1078 | 33 | 7 | 118 | 88 | 12 | 11 | |||
1347 | 15 | 4 | 95 | 155 | 80 | 38 | |||
1418^{+} | 12 | 3 | 98 | 112 | 37 | 11 | |||
1493 | 10 | 2 | 102 | 132 | 56 | 19 | |||
1654 | 11 | 3 | 106 | 155 | 80 | 61 | |||
CPA 2.79 | 70 | 20 | 91 | 842 | 766 | 337 | 44 | 194 to 1457 | |
Key: conc. = Concentration
RTG = Relative Total Growth
SG = Suspension Growth
CE = Cloning Efficiency
MF = Mutant Frequency
IMF = Induced Mutant Frequency
N/A = Not applicable
4NQO = 4-Nitroquinoline
CPA = Cyclophosphamide
* = mean values
^{*} = all reported values from a single culture due to high CE value
The GEF was not exceeded for any concentration tested
Table 3. Osmolality and pH Measurements at the End of Each Treatment Period (Experiment 01) | ||||
Timepoint | Osmolality (mOsm/kg) | pH | ||
3 hour –S9 mix | DMSO | 671.1 µg/mL | DMSO | 671.1 µg/mL |
434 | 420 | 7.27 | 7.28 | |
3 hour +S9 mix | DMSO | 3200 µg/mL | DMSO | 3200 µg/mL |
458 | 431 | 7.21 | 7.21 | |
Table 4. Historical Control Data | ||||||||
Control
| Concentration
| Metabolic Activation
| Treatment
| Range of mutant frequency (within database) minimum to maximum | Mean
| Standard Deviation
| 95% Confidence Limits
| Number of data points |
Solvent (Vehicle) | 1% DMSO | - | 3 h | 62 to 110 | 89 | 15 | 58 to 119 | 16 |
4-Nitroquinoline-1- oxide | 0.001 mM (1 µM) | - | 3 h | 519 to 1181 | 804 | 190 | 431 to 1177 | 16 |
Solvent (Vehicle) | 1% DMSO | + | 3 h | 54 to 113 | 89 | 17 | 55 to 123 | 16 |
Cyclophosphamide | 0.01 mM (10 µM) | + | 3 h | 417 to 1581 | 826 | 322 | 194 to 1457 | 16 |
Data generated: 3h -S9 mix from 25 July 2017 to 26 October 2021
3h +S9 mix from 08 December 2015 to 26 October 2021
Table 1 – Test 1: Petroleum Gas Oil Fraction, Co-pressed with Renewable Hydrocarbons of Plant and/or Animal Origin (EC 941-364-9): plate incorporation method – with metabolic activation
Dose per plate |
Number of revertant colonies per plate |
|||||||||
S. typhimuriumLT2 |
E. coliWP2 |
|||||||||
|
TA1535 |
TA1537 |
TA98 |
TA100 |
uvrA/pKM101 |
|||||
μg |
Mean |
SD |
Mean |
SD |
Mean |
SD |
Mean |
SD |
Mean |
SD |
Solvent control |
12 |
2.1 |
11 |
1.5 |
37 |
2.5 |
138 |
10.0 |
201 |
7.8 |
1.6 |
14 |
2.1 |
13 |
1.2 |
34 |
4.5 |
154 |
5.0 |
202 |
14.6 |
5 |
12 |
2.0 |
12 |
0.6 |
33 |
3.2 |
142 |
11.4 |
201 |
12.4 |
16 |
10 |
1.0 |
13 |
1.5 |
36 |
2.1 |
131 |
7.8 |
214 |
1.5 |
50 |
13 |
2.1 |
11 |
1.5 |
31 |
6.1 |
120 |
11.2 |
187 |
9.5 |
160 |
12 |
2.0 |
12 |
2.1 |
34 |
7.6 |
147 |
6.2 |
191 |
17.9 |
500 |
11 |
3.1 |
9 |
1.2 |
38 |
3.1 |
142 |
18.5 |
192 |
14.7 |
1600 |
11 |
4.7 |
10 |
3.2 |
39 |
2.6 |
150 |
11.4 |
194 |
14.0 |
5000 |
11 |
4.9 |
15 |
1.5 |
40 |
11.4 |
152 |
3.8 |
178 |
17.6 |
Positive control |
176 |
7.5 |
152 |
4.5 |
1586 |
180.4 |
2455 |
20.9 |
1945 |
21.4 |
Positive controls
TA1535: 2-aminoanthracene 2 μg/plate
TA1537: 2-aminoanthracene 2 μg/plate
TA98: 2-aminoanthracene 2 μg/plate
TA100: 2-aminoanthracene 2 μg/plate
uvrA/pKM101: 2-aminoanthracene 20 μg/plate
Table 2 – Test 1: Petroleum Gas Oil Fraction, Co-pressed with Renewable Hydrocarbons of Plant and/or Animal Origin (EC 941-364-9): plate incorporation method – without metabolic activation
Dose per plate |
Number of revertant colonies per plate |
|||||||||
S. typhimuriumLT2 |
E. coliWP2 |
|||||||||
|
TA1535 |
TA1537 |
TA98 |
TA100 |
uvrA/pKM101 |
|||||
μg |
Mean |
SD |
Mean |
SD |
Mean |
SD |
Mean |
SD |
Mean |
SD |
Solvent control |
15 |
1.5 |
11 |
0.6 |
27 |
0.6 |
128 |
2.0 |
170 |
4.2 |
1.6 |
14 |
2.1 |
11 |
2.0 |
28 |
2.0 |
122 |
2.6 |
156 |
10.0 |
5 |
13 |
2.0 |
9 |
3.2 |
25 |
5.6 |
113 |
10.6 |
165 |
2.0 |
16 |
14 |
1.5 |
10 |
0.6 |
24 |
7.1 |
104 |
2.5 |
173 |
3.0 |
50 |
11 |
1.7 |
9 |
3.8 |
23 |
6.1 |
110 |
3.2 |
155 |
4.2 |
160 |
12 |
1.2 |
11 |
1.5 |
23 |
7.0 |
110 |
9.7 |
150 |
13.9 |
500 |
14 |
2.6 |
12 |
1.7 |
26 |
4.5 |
113 |
12.8 |
141 |
8.3 |
1600 |
11 |
0.6 |
10 |
2.5 |
25 |
5.5 |
101 |
10.8 |
141 |
10.0 |
5000 |
10 |
2.0 |
9 |
1.5 |
24 |
2.1 |
100 |
8.2 |
145 |
5.7 |
Positive control |
432 |
7.6 |
195 |
8.3 |
360 |
13.1 |
558 |
28.3 |
1000 |
57.3 |
Positive controls
TA1535: sodium azide 0.5 μg/plate
TA1537: 2-aminocridine.HCl 50 μg/plate
TA98: 2-nitrofluorene 1 μg/plate
TA100: sodium azide 0.5 μg/plate
uvrA/pKM101: potassium dichromate 25 μg/plate
Table 3 – Test 2: Petroleum Gas Oil Fraction, Co-pressed with Renewable Hydrocarbons of Plant and/or Animal Origin (EC 941-364-9): liquid pre-incubation method – with metabolic activation
Dose per plate |
Number of revertant colonies per plate |
|||||||||
S. typhimuriumLT2 |
E. coliWP2 |
|||||||||
|
TA1535 |
TA1537 |
TA98 |
TA100 |
uvrA/pKM101 |
|||||
μg |
Mean |
SD |
Mean |
SD |
Mean |
SD |
Mean |
SD |
Mean |
SD |
Solvent control |
10 |
0.6 |
9 |
1.0 |
32 |
3.1 |
142 |
6.7 |
209 |
6.7 |
1.6 |
11 |
1.0 |
10 |
2.1 |
31 |
2.6 |
148 |
5.0 |
193 |
7.6 |
5 |
12 |
1.0 |
8 |
2.6 |
18 |
8.1 |
140 |
16.6 |
197 |
23.5 |
16 |
10 |
0.6 |
8 |
2.0 |
29 |
6.6 |
115 |
19.3 |
197 |
8.6 |
50 |
10 |
3.1 |
8 |
2.0 |
30 |
6.0 |
120 |
9.2 |
208 |
1.5 |
160 |
9 |
3.1 |
9 |
3.5 |
24 |
2.1 |
113 |
28.7 |
192 |
7.0 |
500 |
10 |
2.1 |
9 |
3.2 |
25 |
5.5 |
125 |
2.1 |
204 |
13.2 |
1600 |
7 |
2.0 |
6 |
2.1 |
25 |
5.6 |
116 |
11.8 |
195 |
12.7 |
5000 |
8 |
1.5 |
8 |
1.5 |
25 |
1.0 |
111 |
11.0 |
175 |
8.5 |
Positive control |
126 |
1.5 |
102 |
6.1 |
1327 |
105.1 |
1769 |
35.4 |
1844 |
56.7 |
Positive controls
TA1535: 2-aminoanthracene 2 μg/plate
TA1537: 2-aminoanthracene 2 μg/plate
TA98: 2-aminoanthracene 2 μg/plate
TA100: 2-aminoanthracene 2 μg/plate
uvrA/pKM101: 2-aminoanthracene 20 μg/plate
Table 4 – Test 2: Petroleum Gas Oil Fraction, Co-pressed with Renewable Hydrocarbons of Plant and/or Animal Origin (EC 941-364-9): liquid pre-incorporation method – without metabolic activation
Dose per plate |
Number of revertant colonies per plate |
|||||||||
S. typhimuriumLT2 |
E. coliWP2 |
|||||||||
|
TA1535 |
TA1537 |
TA98 |
TA100 |
uvrA/pKM101 |
|||||
μg |
Mean |
SD |
Mean |
SD |
Mean |
SD |
Mean |
SD |
Mean |
SD |
Solvent control |
13 |
1.2 |
9 |
0.6 |
28 |
1.2 |
118 |
2.5 |
146 |
7.2 |
1.6 |
12 |
1.0 |
9 |
0.6 |
27 |
5.7 |
120 |
4.2 |
148 |
7.1 |
5 |
12 |
2.0 |
8 |
1.0 |
26 |
1.7 |
113 |
3.5 |
135 |
2.0 |
16 |
11 |
2.6 |
9 |
2.1 |
28 |
2.1 |
116 |
6.8 |
143 |
5.0 |
50 |
10 |
1.5 |
11 |
1.2 |
27 |
4.5 |
110 |
3.6 |
132 |
6.8 |
160 |
11 |
2.1 |
8 |
1.2 |
23 |
6.0 |
101 |
3.0 |
128 |
6.0 |
500 |
10 |
2.6 |
5 |
3.1 |
16 |
1.5 |
98 |
8.9 |
121 |
7.8 |
1600 |
5 |
1.2 |
4 |
0.6 |
17 |
3.1 |
76 |
14.5 |
122 |
17.4 |
5000 |
7 |
1.0 |
3 |
1.5 |
15 |
3.0 |
100 |
7.8 |
100 |
4.0 |
Positive control |
435 |
22.0 |
175 |
15.0 |
343 |
27.4 |
444 |
28.0 |
939 |
29.7 |
Positive controls
TA1535: sodium azide 0.5 μg/plate
TA1537: 2-aminocridine.HCl 50 μg/plate
TA98: 2-nitrofluorene 1 μg/plate
TA100: sodium azide 0.5 μg/plate
uvrA/pKM101: potassium dichromate 25 μg/plate
Table 2. Summary of Analysed Concentrations |
|||||||||
Exposure Condition |
Test Material Concentration (µg/mL) |
RTG%* |
SG* |
CE%* |
X 10-6 |
IMF Small Colonies%* |
(MF) Historical control data 95% confidence limits |
||
MF* |
IMF (pooled all colonies)* |
IMF (small colonies)* |
|||||||
3 hour (-S9 mix) Experiment 02 |
Solvent (DMSO) |
100 |
18 |
111 |
98 |
0 |
0 |
0 |
58 to 111 |
GEF Threshold |
N/A |
N/A |
N/A |
224 |
N/A |
N/A |
N/A |
N/A |
|
39.98 |
101 |
18 |
116 |
83 |
-15 |
1 |
|||
79.97 |
78 |
14 |
111 |
82 |
-16 |
-5 |
|||
98.72 |
57 |
12 |
97 |
101 |
3 |
13 |
|||
109.7 |
32 |
7 |
86 |
103 |
4 |
15 |
|||
121.9 |
25 |
6 |
88 |
133 |
35 |
32 |
|||
135.4 |
11 |
3 |
74 |
188 |
90 |
43 |
|||
150.5 |
8 |
2 |
83 |
168 |
70 |
21 |
|||
4NQO 0.19 |
87 |
18 |
97 |
562 |
464 |
174 |
38 |
445 to 1044 |
|
|
|||||||||
3 hour (+S9 mix) Experiment 02 |
Solvent (DMSO) |
100 |
21 |
108 |
113 |
0 |
0 |
0 |
54 to 115 |
GEF Threshold |
N/A |
N/A |
N/A |
239 |
N/A |
N/A |
N/A |
N/A |
|
38.40 |
101 |
19 |
123 |
87 |
-26 |
-4 |
|||
307.2 |
64 |
14 |
101 |
132 |
19 |
0 |
|||
614.4 |
54 |
12 |
104 |
184 |
71 |
32 |
|||
768.0 |
26 |
7 |
83 |
195 |
82 |
44 |
|||
960.0 |
10 |
3 |
78 |
195 |
82 |
43 |
|||
1200 E |
1 |
1 |
22 |
277 |
164 |
52 |
|||
CPA 2.79 |
52 |
17 |
68 |
1416 |
1303 |
507 |
39 |
165 to 1342 |
|
Key: conc. = Concentration
RTG = Relative Total Growth
SG = Suspension Growth
CE = Cloning Efficiency
MF = Mutant Frequency
IMF = Induced Mutant Frequency
N/A = Not applicable
4NQO = 4-Nitroquinoline
CPA = Cyclophosphamide
* = mean values
E = Excluded as RTG below 10%
GEF = global evaluation factor (126x10-6).
The GEF was not exceeded for any concentration tested
Table 3. Osmolality and pH Measurements at the end of each Treatment (Experiment 01) |
||||
Timepoint |
Osmolality (mOsm/kg) |
pH |
||
3 hour (-S9) |
DMSO |
314.6 µg/mL |
DMSO |
314.6 µg/mL |
433 |
430 |
7.36 |
7.36 |
|
3 hour (+S9) |
DMSO |
1500 µg/mL |
DMSO |
1500 µg/mL |
442 |
439 |
7.26 |
7.29 |
|
Table 4. Historical Control Data |
||||||||
Control |
Concentration |
Metabolic Activation |
Treatment |
Range of Mutant Frequency (within database) Min to Max |
Mean |
Standard Deviation |
95% Confidence Limits |
Number of data points |
Solvent (vehicle) |
1% DMSO |
- |
3h |
62 to 105 |
85 |
14 |
58 to 111 |
13 |
4-Nitroquinoline-1-oxide |
0.001 mM (1 µM) |
- |
3h |
519 to 1069 |
745 |
153 |
445 to 1044 |
13 |
Solvent |
1% DMSO |
+ |
3h |
54 to 112 |
85 |
15 |
54 to 115 |
13 |
Cyclophosphamide |
0.01 mM (10 µM) |
+ |
3h |
417 to 1581 |
753 |
300 |
165 to 1342 |
13 |
Data generated:
3h -S9 mix from 25 July 2017 to 04 May 2021
3h +S9 mix from 08 December 2015 to 04 May 2021
Micronucleus data
Table 1 – Petroleum Gas Oil Fraction, Co-processed with Renewable Hydrocarbons of Plant and/or Animal Origin (EC 941-364-9): 3 hour exposure testing in the presence of S9 mix
Dose (μg/mL) |
3h +S9 treatment |
|||
CBPI |
% Cytostasis |
% MN per dose |
p-value |
|
Solvent (DMSO) |
1.599 |
N/A |
1.0 |
N/A |
296.3 |
1.550 |
8.15 |
1.2 |
ns |
1000 |
1.481 |
19.67 |
1.1 |
ns |
1500P |
1.343 |
42.67 |
1.0 |
ns |
CPA 4 |
1.418 |
30.15 |
4.3 |
2.942×10-11 |
CBPI
= cytokinesis block proliferation index
MN per dose = micronucleated binucleates/2000 binucleates
CPA = cyclophosphamide
P = precipitate observed at the end of the treatment period
ns = not statistically significant (by Fisher’s exact test), % -
individual percentages of micronucleated binucleated cells,Cochran-Armitage
treated test p-value = 8.445×10-1,
therefore not statistically significant
Table 2 – Petroleum Gas Oil Fraction, Co-processed with Renewable Hydrocarbons of Plant and/or Animal Origin (EC 941-364-9): 3 hour exposure testing in the absence of S9 mix
Dose (μg/mL) |
3h +S9 treatment |
|||
CBPI |
% Cytostasis |
% MN per dose |
p-value |
|
Solvent (DMSO) |
1.490 |
N/A |
1.1 |
N/A |
666.7 |
1.426 |
13.05 |
1.3 |
ns |
1000 |
1.315 |
35.63 |
1.1 |
ns |
1500P |
1.231 |
52.84 |
1.0 |
ns |
MMC 50 ng/mL |
1.443 |
9.56 |
4.6 |
1.006×10-11 |
CBPI
= cytokinesis block proliferation index
MN per dose = micronucleated binucleates/2000 binucleates
MMC = mitomycin
P = precipitate observed at the end of the treatment period
ns = not statistically significant (by Fisher’s exact test), % -
individual percentages of micronucleated binucleated cells,Cochran-Armitage
treated test p-value = 5.309×10-1,
therefore not statistically significant
Table 3 – Petroleum Gas Oil Fraction, Co-processed with Renewable Hydrocarbons of Plant and/or Animal Origin (EC 941-364-9): continuous (24 hour) exposure testing in the absence of S9 mix
Dose (μg/mL) |
3h +S9 treatment |
|||
CBPI |
% Cytostasis |
% MN per dose |
p-value |
|
Solvent (DMSO) |
1.460 |
N/A |
1.1 |
N/A |
131.7 |
1.501 |
-8.91 |
1.1 |
ns |
296.3 |
1.335 |
27.10 |
1.0 |
ns |
444.4 |
1.298 |
35.22 |
1.2 |
ns |
666.7 |
1.206 |
55.22 |
1.2 |
ns |
MMC 30 ng/mL |
1.376 |
18.26 |
4.5 |
1.043×10-11 |
COL 7.5 ng/mL |
1.458 |
0.43 |
3.9 |
2.275×10-11 |
CBPI
= cytokinesis block proliferation index
MN per dose = micronucleated binucleates/2000 binucleates
MMC = mitomycin
COL = colchicine
P = precipitate observed at the end of the treatment period
ns = not statistically significant (by Fisher’s exact test), % -
individual percentages of micronucleated binucleated cells,Cochran-Armitage
treated test p-value = 6.801×10-1,
therefore not statistically significant
Cytotoxicity assessment
Table 4 – Petroleum Gas Oil Fraction, Co-processed with Renewable Hydrocarbons of Plant and/or Animal Origin (EC 941-364-9): cytotoxicity assessment in cytokinesis blocked lymphocytes from 3 hour exposure testing in the presence of S9 mix
Concentration |
Number of nucleated cells |
CBPI |
% Cytostasis |
|||
Mono- |
Bi- |
Multi- |
Total scored |
|||
Solvent (DMSO) |
208 |
286 |
7 |
501 |
1.599 |
0.00 |
5.138 |
sns |
sns |
sns |
sns |
ND |
ND |
7.707 |
sns |
sns |
sns |
sns |
ND |
ND |
11.56 |
sns |
sns |
sns |
sns |
ND |
ND |
17.34 |
sns |
sns |
sns |
sns |
ND |
ND |
26.01 |
sns |
sns |
sns |
sns |
ND |
ND |
39.02 |
sns |
sns |
sns |
sns |
ND |
ND |
58.53 |
sns |
sns |
sns |
sns |
ND |
ND |
87.79 |
sns |
sns |
sns |
sns |
ND |
ND |
131.7 |
sns |
sns |
sns |
sns |
ND |
ND |
197.5 |
sns |
sns |
sns |
sns |
ND |
ND |
296.3 |
231 |
263 |
6 |
500 |
1.550 |
8.15 |
444.4 |
250 |
250 |
1 |
501 |
1.503 |
16.00 |
666.7 |
251 |
244 |
5 |
500 |
1.508 |
15.16 |
1000 |
265 |
231 |
5 |
501 |
1.481 |
19.67 |
1500P |
334 |
162 |
5 |
501 |
1.343 |
42.67 |
CPA 4 µg/mL |
316 |
200 |
10 |
526 |
1.418 |
30.15 |
Mono
= mononucleated cells
Bi = binucleated cells
Multi = multinucleated cells
total scored = sum of mono-, bi- and multi-
CBPI = cytokinesis block proliferation index
CPA = cyclophosphamide
sns = slide not scored/analysed
ND = not determined
P = precipitate observed at the end of the treatment period
Table 5 – Petroleum Gas Oil Fraction, Co-processed with Renewable Hydrocarbons of Plant and/or Animal Origin (EC 941-364-9): cytotoxicity assessment in cytokinesis blocked lymphocytes from 3 hour exposure testing in the absence of S9 mix
Concentration µg/mL |
Number of nucleated cells |
CBPI |
% Cytostasis |
|||
Mono- |
Bi- |
Multi- |
Total scored |
|||
Solvent (DMSO) |
283 |
260 |
4 |
547 |
1.490 |
0.00 |
5.138 |
sns |
sns |
sns |
sns |
ND |
ND |
7.707 |
sns |
sns |
sns |
sns |
ND |
ND |
11.56 |
sns |
sns |
sns |
sns |
ND |
ND |
17.34 |
sns |
sns |
sns |
sns |
ND |
ND |
26.01 |
sns |
sns |
sns |
sns |
ND |
ND |
39.02 |
sns |
sns |
sns |
sns |
ND |
ND |
58.53 |
sns |
sns |
sns |
sns |
ND |
ND |
87.79 |
sns |
sns |
sns |
sns |
ND |
ND |
131.7 |
sns |
sns |
sns |
sns |
ND |
ND |
197.5 |
257 |
241 |
3 |
501 |
1.493 |
-0.63 |
296.3 |
274 |
226 |
0 |
500 |
1.452 |
7.74 |
444.4 |
301 |
205 |
0 |
506 |
1.405 |
17.31 |
666.7 |
288 |
211 |
1 |
500 |
1.426 |
13.05 |
1000 |
344 |
156 |
1 |
501 |
1.315 |
35.63 |
1500P |
386 |
116 |
0 |
502 |
1.231 |
52.84 |
MMC 50 ng/mL |
281 |
218 |
2 |
501 |
1.443 |
9.56 |
Mono
= mononucleated cells
Bi = binucleated cells
Multi = multinucleated cells
total scored = sum of mono-, bi- and multi-
CBPI = cytokinesis block proliferation index
MMC = mitomycin C
sns = slide not scored/analysed
ND = not determined
P = precipitate observed at the end of the treatment period
Table 6 – Petroleum Gas Oil Fraction, Co-processed with Renewable Hydrocarbons of Plant and/or Animal Origin (EC 941-364-9): cytotoxicity assessment in cytokinesis blocked lymphocytes from continuous (24 hour) exposure testing in the absence of S9 mix
Concentration |
Number of nucleated cells |
CBPI |
% Cytostasis |
|||
Mono- |
Bi- |
Multi- |
Total scored |
|||
Solvent (DMSO) |
270 |
230 |
0 |
500 |
1.460 |
0.00 |
5.138 |
sns |
sns |
sns |
sns |
ND |
ND |
7.707 |
sns |
sns |
sns |
sns |
ND |
ND |
11.56 |
sns |
sns |
sns |
sns |
ND |
ND |
17.34 |
sns |
sns |
sns |
sns |
ND |
ND |
26.01 |
sns |
sns |
sns |
sns |
ND |
ND |
39.02 |
sns |
sns |
sns |
sns |
ND |
ND |
58.53 |
sns |
sns |
sns |
sns |
ND |
ND |
87.79 |
240 |
259 |
2 |
501 |
1.525 |
-14.12 |
131.7 |
253 |
245 |
3 |
501 |
1.501 |
-8.91 |
197.5 |
325 |
174 |
2 |
501 |
1.355 |
22.76 |
296.3 |
335 |
164 |
2 |
501 |
1.335 |
27.10 |
444.4 |
355 |
141 |
4 |
500 |
1.298 |
35.22 |
666.7 |
397 |
103 |
0 |
500 |
1.206 |
55.22 |
1000 |
399 |
101 |
0 |
500 |
1.202 |
56.09 |
1500P |
450 |
50 |
1 |
501 |
1.104 |
77.44 |
MMC 30 ng/mL |
314 |
184 |
2 |
500 |
1.376 |
18.26 |
Col 7.5 ng/mL |
286 |
199 |
15 |
500 |
1.458 |
0.43 |
Mono
= mononucleated cells
Bi = binucleated cells
Multi = multinucleated cells
total scored = sum of mono-, bi- and multi-
CBPI = cytokinesis block proliferation index
MMC = mitocyin C
sns = slide not scored/analysed
ND = not determined
P = precipitate observed at the end of the treatment period
Endpoint conclusion
- Endpoint conclusion:
- no adverse effect observed (negative)
Genetic toxicity in vivo
Description of key information
Three read-across in-vivo were identified for a structurally related material. In a dominant lethal assay in male mice there was no evidence of heritable genotoxic effects. In a well conducted bone marrow chromosomal aberration assay by the oral route there was no evidence of genotoxic activity. In a further bone marrow chromosomal aberration study by the intraperitoneal route weak activity was observed. Effects were only seen at high dose levels (2.0 and 6.0 ml/kg) and the magnitude of effect was small and restricted to chromosomal fragments.
Endpoint conclusion
- Endpoint conclusion:
- no adverse effect observed (negative)
Additional information
Additional information from genetic
toxicity in vivo:
Based on read-across to data on structurally related materials, the substance is not considered to be genotoxic. There was no evidence of activity in two in-vitro assays and two out of three in-vivo studies. In one chromosomal aberration study, weak activity was seen following intraperitoneal treatment at high doses. The magnitude of effects was small and was restricted to small increases in chromosomal fragments.
Justification for selection of
genetic toxicity endpoint
key in-vivo study, selected from 6 in-vitro and 3 in-vivo assays.
Justification for classification or non-classification
The three in vitro assays conducted on EC 941 -803 -4 resulted in negative outcomes. Based on the current data available from EC 941 -803 -4 and read across substances , EC 941 -803 -4 is not classified as mutagenic.
Some oil products containing relatively high concentrations of polycyclic aromatic compounds (PAC) are considered genotoxic carcinogens, and, consequently, are classified and labelled as carcinogenic, Cat. 1A or 1B (H350) or Cat. 2 (H351) according to the EU CLP Regulation (EC) 1272/2008. This classification as carcinogenic does not automatically imply that these substances need also to be classified as mutagenic as defined by the CLP Regulation. The EU legislation aims primarily to classify substances as mutagenic if there is evidence of producing heritable genetic damage, i.e. evidence of producing mutations that are transmitted to the progeny or evidence of producing somatic mutations in combination with evidence of the substance or relevant metabolite reaching the germ line cells in the reproductive organs. The PAC in oil products are poorly bioavailable due to their physico-chemical properties (low water solubility and high molecular weight), making it unlikely that the genotoxic constituents can reach and cause damage to germ cells (Roy, 2007; Potter, 1999). Considering their poor bioavailability, oil products which have been classified as carcinogenic do not need to be classified as mutagenic unless there is clear evidence that germ cells are affected by exposure, consistent with the CLP Regulation. For example, based on in vivo micronucleus tests on home heating oil as well as for read-across substances that were all negative for genotoxicity, vacuum distillate fuels are not classified as mutagens according to the EU CLP Regulation (EC) 1272/2008.
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